Ketol-acid reductoisomerase using NADH

ABSTRACT

Methods for the evolution of NADPH specific ketol-acid reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from  Pseudomonas  that have undergone co-factor switching to utilize NADH are described.

This application is a continuation-in-part of U.S. Ser. No. 12/337,736, filed Dec. 18, 2008, now U.S. Pat. No. 8,129,162, and claims the benefit of the U.S. Provisional Applications 61/015,346, filed Dec. 20, 2007, and 61/109,297, filed Oct. 29, 2008.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCII text file (Name: CL4165USCIP_Corrected_SequenceListing_ascii.txt; Size: 218 Kbs; and Date of Creation: (Nov. 5, 2013)) is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to protein evolution. Specifically, ketol-acid reductoisomerase enzymes have been evolved to use the cofactor NADH instead of NADPH.

BACKGROUND OF THE INVENTION

Ketol-acid reductoisomerase enzymes are ubiquitous in nature and are involved in the production of valine and isoleucine, pathways that may affect the biological synthesis of isobutanol. Isobutanol is specifically produced from catabolism of L-valine as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of incomplete metabolism of amino acids by yeasts. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the Ehrlich pathway (Dickinson, et al., J. Biol. Chem., 273: 25752-25756, 1998).

Addition of exogenous L-valine to the fermentation increases the yield of isobutanol, as described by Dickinson et al., supra, wherein it is reported that a yield of isobutanol of 3 g/L is obtained by providing L-valine at a concentration of 20 g/L in the fermentation. In addition, production of n-propanol, isobutanol and isoamylalcohol has been shown by calcium alginate immobilized cells of Zymomonas mobilis (Oaxaca, et al., Acta Biotechnol., 11: 523-532, 1991).

An increase in the yield of C3-C5 alcohols from carbohydrates was shown when amino acids leucine, isoleucine, and/or valine were added to the growth medium as the nitrogen source (WO 2005040392).

While methods described above indicate the potential of isobutanol production via biological means these methods are cost prohibitive for industrial scale isobutanol production. The biosynthesis of isobutanol directly from sugars would be economically viable and would represent an advance in the art. However, to date the only ketol-acid reductoisomerase (KARI) enzymes known are those that bind NADPH in its native form, reducing the energy efficiency of the pathway. A KARI that would bind NADH would be beneficial and enhance the productivity of the isobutanol biosynthetic pathway by capitalizing on the NADH produced by the existing glycolytic and other metabolic pathways in most commonly used microbial cells. The discovery of a KARI enzyme that can use NADH as a cofactor as opposed to NADPH would be an advance in the art.

The evolution of enzymes having specificity for the NADH cofactor as opposed to NADPH is known for some enzymes and is commonly referred to as “cofactor switching”. See for example Eppink, et al. (J. Mol. Biol., 292: 87-96, 1999), describing the switching of the cofactor specificity of strictly NADPH-dependent p-Hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens by site-directed mutagenesis; and Nakanishi, et al., (J. Biol. Chem., 272: 2218-2222, 1997), describing the use of site-directed mutagenesis on a mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D) resulting in an enzyme having a 200-fold increase in the K_(M) values for NADP(H) and a corresponding decrease of more than 7-fold in those for NAD(H). Co-factor switching has been applied to a variety of enzymes including monooxygenases, (Kamerbeek, et al., Eur. J, Biochem., 271: 2107-2116, 2004); dehydrogenases; Nishiyama, et al., J. Biol. Chem., 268: 4656-4660, 1993; Ferredoxin-NADP reductase, Martinez-Julvez, et al., Biophys. Chem., 115: 219-224, 2005); and oxidoreductases (US2004/0248250).

Rane et al., (Arch. Biochem. Biophys., 338: 83-89, 1997) discuss cofactor switching of a ketol acid reductoisomerase isolated from E. coli by targeting four residues in the enzyme for mutagenesis, (R68, K69, K75, and R76,); however the effectiveness of this method is in doubt.

Although the above cited methods suggest that it is generally possible to switch the cofactor specificity between NADH and NADPH, the methods are enzyme specific and the outcomes unpredictable. The development of a ketol-acid reductoisomerase having a high specificity for NADH with decreased specificity for NADPH would greatly enhance this enzyme's effectiveness in the isobutanol biosynthetic pathway and hence increase isobutanol production. However, no such KARI enzyme has been reported.

SUMMARY OF THE INVENTION

Applicants have solved the stated problem by identifying a number of mutant ketol-acid reductoisomerase enzymes that either have a preference for specificity for NADH as opposed to NADPH or use NADH exclusively in their reaction. The method involves mutagenesis of certain specific residues in the KARI enzyme to produce the co-factor switching.

Accordingly the invention provides A mutant ketol-acid reductoisomerase enzyme comprising the amino acid sequence as set forth in SEQ ID NO: 29; a nucleic acid molecule encoding a mutant ketol-acid reductoisomerase enzyme having the amino acid sequence as set forth in SEQ ID NO:19; a mutant ketol-acid reductoisomerase enzyme as set for in SEQ ID NO:19; a mutant ketol-acid reductoisomerase enzyme having the amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 67, 68, 70, 75, 79, 80, 81 and 82; and a mutant ketol-acid reductoisomerase enzyme as set forth in SEQ ID NO:17 comprising at least one mutation at a residue selected from the group consisting of 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, 165, and 170.

In another embodiment the invention provides a method for the evolution of an NADPH binding ketol-acid reductoisomerase enzyme to an NADH using form comprising:

-   -   a) providing a ketol-acid reductoisomerase enzyme which uses         NADPH having a specific native amino acid sequence;     -   b) identifying the cofactor switching residues in the enzyme         of (a) based on the amino acid sequence of the Pseudomonas         fluorescens ketol-acid reductoisomerase enzyme as set for the in         SEQ ID NO:17 wherein the cofactor switching residues are at         positions selected from the group consisting of: 24, 33, 47, 50,         52, 53, 61, 80, 115, 156, 165, and 170; and     -   c) creating mutations in at least one of the cofactor switching         residues of (b) to create a mutant enzyme wherein said mutant         enzyme binds NADH.

In another embodiment the invention provides a method for the production of isobutanol comprising:

-   -   a) providing a recombinant microbial host cell comprising the         following genetic constructs:         -   i) at least one genetic construct encoding an acetolactate             synthase enzyme for the conversion of pyruvate to             acetolactate;         -   ii) at least one genetic construct encoding a ketol-acid             reductoisomerase enzyme of either of Claims 1 or 6;         -   iii) at least one genetic construct encoding an acetohydroxy             acid dehydratase for the conversion of             2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step             c);         -   iv) at least one genetic construct encoding a branched-chain             keto acid decarboxylase, of the conversion of             α-ketoisovalerate to isobutyraldehyde, (pathway step d);         -   v) at least one genetic construct encoding a branched-chain             alcohol dehydrogenase for the conversion of isobutyraldehyde             to isobutanol (pathway step e); and     -   b) growing the host cell of (a) under conditions where         iso-butanol is produced.

In another embodiment the invention provides a method for the evolution and identification of an NADPH binding ketol-acid reductoisomerase enzyme to an NADH using form comprising:

-   -   a) providing a ketol-acid reductoisomerase enzyme which uses         NADPH having a specific native amino acid sequence;     -   b) identifying the amino acid residues in the native amino acid         sequence whose side chains are in close proximity to the         adenosyl 2′-phosphate of NADPH as mutagenesis targets;     -   c) creating a library of mutant ketol-acid reductoisomerase         enzymes from the class I ketol-acid reductoisomerase enzyme of         step (a), having at least one mutation in at least one of the         mutagenesis target sites of step (b); and     -   d) screening the library of mutant ketol-acid reductoisomerase         enzymes of step (c) to identify NADH binding mutant of         ketol-acid reductoisomerase enzyme.

Alternatively the invention provides a method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising:

-   -   a) providing a mutant enzyme having an amino acid sequence         selected from the group consisting of SEQ ID NOs: 28, 67, 68,         69, 70, and 84;     -   b) constructing a site-saturation library targeting amino acid         positions 47, 50, 52 and 53 of the mutant enzyme of (a); and     -   c) screening the site-saturation library of (b) to identify         mutants which accept NADH instead of NADPH as cofactor.

Similarly the invention provides a method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising:

-   -   a) providing a DNA fragment encoding a mutant enzyme having an         amino acid sequence selected from the group consisting of SEQ ID         NOs: 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98         containing mutations in cofactor specificity domain;     -   b) producing a DNA fragment cofactor specificity domain of (a);     -   c) providing a DNA fragment encoding a mutant enzyme having         mutations in cofactor binding affinity domain selected from the         group consisting of SEQ ID NOs: 28, 67, 68, 69, 70, 84 and 86;     -   d) incorporating mutations of step (b) into mutants of step (c);         and     -   e) screening mutants of step (d) for mutant enzymes having a         ratio of NADH/NADPH utilization is greater than one.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detailed description, the Figures, and the accompanying sequence descriptions, which form part of this application.

FIGS. 1A and 1B—Show four different isobutanol biosynthetic pathways. The steps labeled “a”, “b”, “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j” and “k” represent the substrate to product conversions described below.

FIGS. 2A and 2B—Multiple sequence alignment (MSA) of KARI enzymes from different resources; FIG. 2A—MSA among three NADPH-requiring KARI enzymes: residues 44-56 of SEQ ID NO: 17, residues 44-56 of SEQ ID NO: 16, and residues 88-100 of SEQ ID NO: 18; FIG. 2B—MSA among PF5-KARI and other KARI enzymes, with promiscuous nucleotide specificity, where, MMC5—is from Methanococcus maripaludis C5 (residues 44-57 of SEQ ID NO: 9); MMS2—is from Methanococcus maripaludis S2 (residues 44-57 of SEQ ID NO: 10); MVSB—is from Methanococcus vannielii SB (residues 44-57 of SEQ ID NO: 11); PF5-ilvC—is from Pseudomonas fluorescens (residues 44-57 of SEQ ID NO: 17); ilv5—is from Saccharomyces cerevisiae ilv5 (residues 59-72 of SEQ ID NO: 12); KARI-D1—is from Sulfolobus solfataricus P2 ilvC (residues 48-61 of SEQ ID NO: 13); KARI-D2—is from Pyrobaculum aerophilum str. IM2 ilvC (residues 45-58 of SEQ ID NO: 14); and KARI S1—is from Ralstonia solanacearum GMI1000 ilvC (residues 44-57 of SEQ ID NO: 15). The Consensus sequence is SEQ ID NO: 99.

FIG. 3—Interaction of phosphate binding loop with NADPH based on homology modeling.

FIG. 4—KARI activities of top performers from library C using cofactor NADH versus NADPH. Activity and standard deviation were derived from triple experiments. The mutation information is as follows: C3A7=R47Y/S50A/T52D/V53W; C3A10=R47Y/S50A/T52G/V53W; C3B11=R47F/S50A/T52D/V53W; C3C8=R47G/S50M/T52D/V53W; and C4D12=R47C/S50MT52D/V53W

FIGS. 5A and 5B—FIG. 5A—Comparison of KARI activities of top performers from libraries E, F and G using cofactors NADH and NADPH. FIG. 5B—KARI activities of positive control versus wild type Pf5-ilvC using cofactors NADH. Activity and standard deviation were derived from at least three parallel experiments. “Wt” represents the wild type of Pf5-ilvC and “Neg” means negative control. Experiments for NADH and NADPH reactions in FIG. 5A were 30 min; in FIG. 5B were 10 min.

FIG. 6—Activities of top performers from library H using cofactors NADH versus NADPH. Activity and standard deviation were derived from triple experiments. Mutation information is as follows: 24F9=R47P/S50G/T52D; 68F10=R47P/T52S; 83G10=R47P/S50D/T52S; 39G4=R47P/S50C/T52D; 91A9=R47P/S50CT52D; and C3B11=R47F/S50A/T52D/V53W and Wt is wild type.

FIG. 7—Thermostability of wild type PF5-ilvC. The remaining activity of the enzyme after heating at certain temperatures for 10 min was the average number of triple experiments and normalized to the activity measured at room temperature.

FIG. 8—Multiple DNA sequence alignment among 5 naturally existing KARI molecules: residues 18-193 of SEQ ID NO: 47, residues 17-192 of SEQ ID NO: 48, residues 122-139 of SEQ ID NO: 18, residues 17-193 of SEQ ID NO: 16, and residues 17-193 of SEQ ID NO: 17. The positions both bolded and boxed were identified by error prone PCR and the positions only boxed were targeted for mutagenesis.

FIGS. 9A through 9K—Alignment of the twenty-four functionally verified KARI sequences. The GxGXX(G/A) motif (SEQ ID NO: 100) involved in the binding of NAD(P)H is indicated below the alignment.

FIGS. 10A and 10B—An example of the alignment of Pseudomonas fluorescens Pf-5 KARI to the profile HMM of KARI. The uppermost sequence is SEQ ID NO: 101 and the bottom sequence is SEQ ID NO: 17. The eleven positions that are responsible for co-factor switching are boxed.

FIG. 11—(A) is a linear depiction of the KARI amino acid sequence with specific amino acids numbered. The cofactor specificity domain residues are shown in shaded rectangles. The cofactor binding domain is shown in dotted ovals. (Table A) shows changed amino acids, using single letter code, at numbered positions in four KARI mutants.

FIG. 12 (A) is a linear depiction of the KARI amino acid sequence with specific amino acids numbered. The cofactor specificity domain residues are shown in shaded rectangles. (B) Depicts the first PCR step amplifying the mutated cofactor specificity domain residues. (C) is a linear depiction of the KARI amino acid sequence with specific amino acids of the cofactor binding domain shown in dotted ovals. (D) Depicts incorporation of the domain swapping library into the mutants containing K_(M) improving mutations. Table (E) summaries the K_(M) values for NADH for mutations resulting from combining mutations in the cofactor binding affinity domain with mutations in the cofactor specificity determining domain.

Table 9—is a table of the Profile HMM of the KARI enzymes described in Example 3. The eleven positions in the profile HMM representing the columns in the alignment which correspond to the eleven cofactor switching positions in Pseudomonas fluorescens Pf-5 KARI are identified as positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170. The lines corresponding to these positions in the model file are highlighted in yellow. Table 9 is submitted herewith electronically and is incorporated herein by reference.

The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with the World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

TABLE 1 Oligonucleotide Primers Used In This Invention SEQUENCE ID No. SEQUENCE Description 1 TGATGAACATCTTCGCGTATTCGCCGTCCT Reverse Primer for pBAD vector 2 GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCNN Forward primer GGCTNNCTGGGCCAAGGCT GAAGCCCACGGCTTG library C 3 GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCTCG Forward primer for GCTACCGTTGCCAAGGCTGAAGCCCACGGCTTG library E 4 GCGTAGACGTGACTGTTGGCCTGCGTAAAGGCNNT Forward primer for GCTACCGTTGCCAAGGCTGAAGCCCACGGCTTG library F 5 GCGTAGACGTGACTGTTGGCCTGCGTAAAGGCTCG Forward primer for GCTNNTGTTGCCAAGGCTGAAGCCCACGGCTTG library G 6 GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCNNT Forward primer for GCTNNTGTTGCCAAGGCTGAAGCCCACGGCTTG library H 7 AAGATTAGCGGATCCTACCT Sequencing primer (forward) 8 AACAGCCAAGCTTTTAGTTC Sequencing primer (reverse) 20 CTCTCTACTGTTTCTCCATACCCG pBAD_266-021308f 21 CAAGCCGTGGGCTTCAGCCTTGGCKNN PF5_53Mt022908r 22 CGGTTTCAGTCTCGTCCTTGAAG pBAD_866-021308 49 GCTCAAGCANNKAACCTGAAGG pBAD-405- C33_090808f 50 CCTTCAGGTTKNNTGCTTGAGC pBAD-427- C33_090808r 51 GTAGACGTGNNKGTTGGCCTG pBAD-435- T43_090808f 52 CAGGCCAACKNNCACGTCTAC pBAD-456- T43_090808r 53 CTGAAGCCNNKGGCNNKAAAGTGAC pBAD-484- H59L61_090808f 54 GTCACTTTKNNGCCKNNGGCTTCAG pBAD-509- H59L61_090808r 55 GCAGCCGTTNNKGGTGCCGACT pBAD-519- A71_090808f 56 AGTCGGCACCKNNAACGGCTGC pBAD-541- A71_090808r 57 CATGATCCTGNNKCCGGACGAG pBAD-545- T80_090808f 58 CTCGTCCGGKNNCAGGATCATG pBAD-567- T80_090808r 59 CAAGAAGGGCNNKACTCTGGCCT pBAD-608- A101_090808f 60 AGGCCAGAGTKNNGCCCTTCTTG pBAD-631- A101_090808r 61 GTTGTGCCTNNKGCCGACCTCG pBAD-663- R119_090808f 62 CGAGGTCGGCKNNAGGCACAAC pBAD-685- R119_090808r 71 GTAGACGTGACTGTTGGCCTGNNKAAAGGCNNKGC PF5_4Mt111008.f TNNKNNKGCCAAGGCTGAAGCCCACGG 72 CCGTGGGCTTCAGCCTTGGCKNNKNNAGCKNNGC PF5_4Mt111008.r CTTTKNNCAGGCCAACAGTCACGTCTAC 73 AAGATTAGCGGATCCTACCT pBAD_230.f 74 GAGTGGCGCCCTTCTTGATGTTCG pBAD_601_021308r

Additional sequences used in the application are listed below. The abbreviated gene names in bracket are used in this disclosure.

SEQ ID NO: 9—Methanococcus maripaludis C5-ilvC (MMC5)—GenBank Accession Number NC_(—)009135.1 Region: 901034 . . . 902026

SEQ ID NO: 10 is the Methanococcus maripaludis S2-ilvC (MMS2)—GenBank Accession Number NC_(—)005791.1 Region: 645729 . . . 646721

SEQ ID NO: 11 is the Methanococcus vannielii SB-ilv5 (MVSB)—GenBank Accession Number NZ_AAWX01000002.1 Region: 302214 . . . 303206

SEQ ID NO: 12 is the Saccharomyces cerevisiae ilv5 (ilv5)—GenBank Accession Number NC_(—)001144.4 Region: 838065 . . . 839252

SEQ ID NO: 13 is the Sulfolobus solfataricus P2 ilvC (KARI-D1)—GenBank Accession Number NC_(—)002754.1 Region: 506253 . . . 507260

SEQ ID NO: 14 is the Pyrobaculum aerophilum str. IM2 ilvC (KARI-D2)—GenBank Accession Number NC_(—)003364.1 Region: 1976281 . . . 1977267

SEQ ID NO: 15 is the Ralstonia solanacearum GMI1000 ilvC (KARI-S1)—GenBank Accession Number NC_(—)003295.1 Region: 2248264 . . . 2249280

SEQ ID NO: 16 is the Pseudomonas aeruginosa PAO1 ilvC—GenBank Accession Number NC_(—)002516 Region: 5272455 . . . 5273471

SEQ ID NO: 17 is the Pseudomonas fluorescens PF5 ilvC—GenBank Accession Number NC_(—)004129 Region: 6017379 . . . 6018395

SEQ ID NO: 18 is the Spinacia oleracea ilvC (Spinach-KARI)—GenBank Accession Number NC_(—)002516 Region: 1 . . . 2050.

SEQ ID NO: 19 is the amino acid sequence of the mutant (Y24F/R47Y/S50A/T52D/V53A/L61F/G170A) of the ilvC native protein of Pseudomonas fluorescens.

SEQ ID NO: 24 is the amino acid SEQ of the mutant ZB1 (Y24F/R47Y/S50A/T52D/V53A/L61F/A156V)

SEQ ID NO: 25 is the amino acid SEQ of the mutant ZF3 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F)

SEQ ID NO: 26 is the amino acid SEQ of the mutant ZF2 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/A156V)

SEQ ID NO: 27 is the Amino Acid SEQ of the Mutant Zb3 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/G170A)

SEQ ID NO: 28 is the amino acid SEQ of the mutant Z4B8 (C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V/G170A)

SEQ ID NO: 29 is a consensus amino acid sequence comprising all experimentally verified KARI point mutations as based on SEQ ID NO:17.

SEQ ID NO: 30 is the amino acid sequence for KARI from Natronomonas pharaonis DSM 2160

SEQ ID NO: 31 is the amino acid sequence for KARI from Bacillus subtilis subsp. subtilis str. 168

SEQ ID NO: 32 is the amino acid sequence for KARI from Corynebacterium glutamicum ATCC13032

SEQ ID NO: 33 is the amino acid sequence for KARI from Phaeospirilum molischianum

SEQ ID NO: 34 is the amino acid sequence for KARI from Zymomonas mobilis subsp. mobilis ZM4

SEQ ID NO: 35 is the amino acid sequence for KARI Alkalilimnicola ehrlichei MLHE-1

SEQ ID NO: 36 is the amino acid sequence for KARI from Campylobacter lari RM2100

SEQ ID NO: 37 is the amino acid sequence for KARI from Marinobacter aquaeolei VT8

SEQ ID NO: 38 is the amino acid sequence for KARI Psychrobacter arcticus 273-4

SEQ ID NO: 39 is the amino acid sequence for KARI from Hahella chejuensis KCTC2396

SEQ ID NO: 40 is the amino acid sequence for KARI from Thiobacillus denitrificans ATCC25259

SEQ ID NO: 41 is the amino acid sequence for KARI from Azotobacter vinelandii AvOP

SEQ ID NO: 42 is the amino acid sequence for KARI from Pseudomonas syringae pv. syringae B728a

SEQ ID NO: 43 is the amino acid sequence for KARI from Pseudomonas syringae pv. tomato str. DC3000

SEQ ID NO: 44 is the amino acid sequence for KARI from Pseudomonas putida KT2440

SEQ ID NO: 45 is the amino acid sequence for KARI from Pseudomonas entomophila L48

SEQ ID NO: 46 is the amino acid sequence for KARI from Pseudomonas mendocina ymp

SEQ ID NO: 47 is the amino acid sequence for KARI from Bacillus cereus ATCC10987 NP_(—)977840.1

SEQ ID NO: 48 is the amino acid sequence for KARI from Bacillus cereus ATCC10987 NP_(—)978252.1

SEQ ID NO: 63 is the amino acid sequence for KARI from Escherichia coli—GenBank Accession Number P05793

SEQ ID NO: 64 is the amino acid sequence for KARI from Marine Gamma Proteobacterium HTCC2207—GenBank Accession Number ZP_(—)01224863.1

SEQ ID NO: 65 is the amino acid sequence for KARI from Desulfuromonas acetoxidans—GenBank Accession Number ZP_(—)01313517.1

SEQ ID NO: 66 is the amino acid sequence for KARI from Pisum sativum (Pea)—GenBank Accession Number O82043

SEQ ID NO: 67 is the amino acid sequence for mutant 3361G8 (C33L/R47Y/S50A/T52D/V53A/L61F/T80I)

SEQ ID NO: 68 is the amino acid sequence for mutant 2H10 (Y24F/C33L/R47Y/S50A/T52D/V53I/L61F/T80I/A156V)

SEQ ID NO: 69 is the amino acid sequence for mutant 1D2 (Y24F/R47Y/S50A/T52D/V53A/L61F/T80I/A156V.

SEQ ID NO: 70 is the amino acid sequence for mutant 3F12 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V).

SEQ ID NO: 75 is the amino acid sequence for mutant JB1C6 (Y24F/C33L/R47H/S50D/T52Y/V53Y/L61F/T80I/A156V)

SEQ ID NO: 76 is the amino acid sequence for mutant 16445E4 (C33L/R47P/S50V/T52D/V53G/L61F/T80I/A156V)

SEQ ID NO: 77 is the amino acid sequence for mutant 16468D7 (Y24F/C33L/R47T/S50I/T52D/V53R/L61F/T80I/A156V)

SEQ ID NO: 78 is the amino acid sequence for mutant 16469F3 (C33L/R47E/S50A/T52D/V53A/L61F/T80I)

SEQ ID NO: 79 is the Amino Acid Sequence for Mutant JEA1 (Y24F/C33L/R47P/S50F/T52D/L61F/T80I/A156V)

SEQ ID NO: 80 is the amino acid sequence for mutant JEG2 (Y24F/C33L/R47F/S50A/T52D/V53A/L61F/T80I/A156V)

SEQ ID NO: 81 is the amino acid sequence for mutant JEG4 (Y24F/C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)

SEQ ID NO: 82 is the amino acid sequence for mutant JEA7 (Y24F/C33L/R47P/S50N/T52D/V53A/L61F/T80I/A156V)

SEQ ID NO: 83 is the amino acid sequence for mutant JED1 (C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)

SEQ ID NO: 84 is the Amino Acid Sequence for Mutant 3361E1

SEQ ID NO: 85 is the amino acid sequence for mutant C2F6

SEQ ID NO: 86 is the amino acid sequence for mutant C3B11

SEQ ID NO: 87 is the amino acid sequence for mutant C4D12

SEQ ID NO: 88 is the amino acid sequence for mutant SE1

SEQ ID NO: 89 is the amino acid sequence for mutant SE2

SEQ ID NO: 90 is the amino acid sequence for mutant SB3

SEQ ID NO: 91 is the amino acid sequence for mutant SD3

SEQ ID NO: 92 is the amino acid sequence for mutant 9650E5

SEQ ID NO: 93 is the amino acid sequence for mutant 9667A11

SEQ ID NO: 94 is the amino acid sequence for mutant 9862B9

SEQ ID NO: 95 is the amino acid sequence for mutant 9875B9

SEQ ID NO: 96 is the amino acid sequence for mutant 11461D8

SEQ ID NO: 97 is the amino acid sequence for mutant 11463

SEQ ID NO: 98 is the amino acid sequence for mutant 11518B4

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the generation of mutated KARI enzymes to use NADH as opposed to NADPH. Such co-factor switched enzymes function more effectively in microbial systems designed to produce isobutanol. Isobutanol is an important industrial commodity chemical with a variety of applications, where its potential as a fuel or fuel additive is particularly significant. Although only a four-carbon alcohol, butanol has the energy content similar to that of gasoline and can be blended with any fossil fuel. Isobutanol is favored as a fuel or fuel additive as it yields only CO₂ and little or no SO_(x) or NO_(x) when burned in the standard internal combustion engine. Additionally butanol is less corrosive than ethanol, the most preferred fuel additive to date.

The following definitions and abbreviations are to be use for the interpretation of the claims and the specification.

The term “invention” or “present invention” as used herein is meant to apply generally to all embodiments of the invention as described in the claims as presented or as later amended and supplemented, or in the specification.

The term “isobutanol biosynthetic pathway” refers to the enzymatic pathway to produce isobutanol. Preferred isobutanol biosynthetic pathways are illustrated in FIG. 1 and described herein.

The term “NADPH consumption assay” refers to an enzyme assay for the determination of the specific activity of the KARI enzyme, involving measuring the disappearance of the KARI cofactor, NADPH, from the enzyme reaction.

“KARI” is the abbreviation for the enzyme ketol-acid reductoisomerase.

The term “close proximity” when referring to the position of various amino acid residues of a KARI enzyme with respect to the adenosyl 2′-phosphate of NADPH means amino acids in the three-dimensional model for the structure of the enzyme that are within about 4.5 Å of the phosphorus atom of the adenosyl 2′-phosphate of NADPH bound to the enzyme.

The term “ketol-acid reductoisomerase” (abbreviated “KARI”), and “acetohydroxy acid isomeroreductase” will be used interchangeably and refer to the enzyme having the EC number, EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego). Ketol-acid reductoisomerase catalyzes the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate, as more fully described below. These enzymes are available from a number of sources, including, but not limited to E. coli GenBank Accession Number NC-000913 REGION: 3955993 . . . 3957468, Vibrio cholerae GenBank Accession Number NC-002505 REGION: 157441 . . . 158925, Pseudomonas aeruginosa, GenBank Accession Number NC-002516, (SEQ ID NO: 16) REGION: 5272455 . . . 5273471, and Pseudomonas fluorescens GenBank Accession Number NC-004129 (SEQ ID NO: 17) REGION: 6017379 . . . 6018395. As used herein the term “Class I ketol-acid reductoisomerase enzyme” means the short form that typically has between 330 and 340 amino acid residues, and is distinct from the long form, called class II, that typically has approximately 490 residues.

The term “acetolactate synthase” refers to an enzyme that catalyzes the conversion of pyruvate to acetolactate and CO₂. Acetolactate has two stereoisomers ((R) and (S)); the enzyme prefers the (S)-isomer, which is made by biological systems. Preferred acetolactate synthases are known by the EC number 2.2.1.6 9 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB15618, Z99122, NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), Klebsiella pneumoniae (GenBank Nos: AAA25079, M73842 and Lactococcus lactis (GenBank Nos: AAA25161, L16975).

The term “acetohydroxy acid dehydratase” refers to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoiso-valerate. Preferred acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. These enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP_(—)026248, NC_(—)000913, S. cerevisiae (GenBank Nos: NP_(—)012550, NC_(—)001142), M. maripaludis (GenBank Nos: CAF29874, BX957219), and B. subtilis (GenBank Nos: CAB14105, Z99115).

The term “branched-chain α-keto acid decarboxylase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde and CO₂. Preferred branched-chain α-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166, AY548760; CAG34226, AJ746364, Salmonella typhimurium (GenBank Nos: NP-461346, NC-003197), and Clostridium acetobutylicum (GenBank Nos: NP-149189, NC-001988).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Preferred branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH (reduced nicotinamide adenine dinucleotide) and/or NADPH as electron donor and are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos: NP-010656, NC-001136; NP-014051, NC-001145), E. coli (GenBank Nos: NP-417484, and C. acetobutylicum (GenBank Nos: NP-349892, NC_(—)003030).

The term “branched-chain keto acid dehydrogenase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyryl-CoA (isobutyryl-cofactor A), using NAD⁺ (nicotinamide adenine dinucleotide) as electron acceptor. Preferred branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. These branched-chain keto acid dehydrogenases comprise four subunits, and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, B. subtilis (GenBank Nos: CAB14336, Z99116; CAB14335, Z99116; CAB14334, Z99116; and CAB14337, Z99116) and Pseudomonas putida (GenBank Nos: AAA65614, M57613; AAA65615, M57613; AAA65617, M57613; and AAA65618, M57613).

The terms “k_(cat)” and “K_(M)” are known to those skilled in the art and are described in Enzyme Structure and Mechanism, 2^(nd) ed. (Ferst; W.H. Freeman Press, NY, 1985; pp 98-120). The term “k_(cat)”, often called the “turnover number”, is defined as the maximum number of substrate molecules converted to products per active site per unit time, or the number of times the enzyme turns over per unit time. K_(cat)=Vmax/[E], where [E] is the enzyme concentration (Ferst, supra). The terms “total turnover” and “total turnover number” are used herein to refer to the amount of product formed by the reaction of a KARI enzyme with substrate.

The term “catalytic efficiency” is defined as the K_(cat)/K_(M) of an enzyme. Catalytic efficiency is used to quantify the specificity of an enzyme for a substrate.

The term “isolated nucleic acid molecule”, “isolated nucleic acid fragment” and “genetic construct” will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of a microorganism. A “foreign” gene refers to a gene not normally found in the host microorganism, but that is introduced into the host microorganism by gene transfer. Foreign genes can comprise native genes inserted into a non-native microorganism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

As used herein the term “coding sequence” refers to a DNA sequence that encodes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host microorganism, resulting in genetically stable inheritance. Host microorganisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” microorganisms.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

The term “site-saturation library” refers to a library which contains random substitutions at a specific amino acid position with all 20 possible amino acids at once.

The term “error-prone PCR” refers to adding random copying errors by imposing imperfect or ‘sloppy’ PCR reaction conditions which generate randomized libraries of mutations in a specific nucleotide sequence.

As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host microorganism without altering the polypeptide encoded by the DNA.

Molecular Techniques

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook et al. (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) (hereinafter “Maniatis”); and by Silhavy et al. (Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y., 1984); and by Ausubel, F. M. et al., (Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience, 1987).

The present invention addresses a need that arises in the microbial production of isobutanol where the ketol-acid reductoisomerase enzyme performs a vital role. Wild type ketol-acid reductoisomerase enzymes typically use NADPH as their cofactor. However, in the formation of isobutanol an excess of NADH is produced by ancillary metabolic pathways. The invention provides mutant Class I KARI enzymes that have been evolved to utilize NADH as a cofactor, overcoming the cofactor problem and increasing the efficiency of the isobutanol biosynthetic pathway.

Production of isobutanol utilizes the glycolysis pathway present in the host microorganism. During the production of two molecules of pyruvate from glucose during glycolysis, there is net production of two molecules of NADH from NAD⁺ by the glyceraldehyde-3-phosphate dehydrogenase reaction. During the further production of one molecule of isobutanol from two molecules of pyruvate, there is net consumption of one molecule of NADPH, by the KARI reaction, and one molecule of NADH by the isobutanol dehydrogenase reaction. The overall reaction of glucose to isobutanol thus leads to net production of one molecule of NADH and net consumption of one molecule of NADPH. The interconversion of NADH with NADPH is generally slow and inefficient; thus, the NADPH consumed is generated by metabolism (for example, by the pentose phosphate pathway) consuming substrate in the process. Meanwhile, the cell strives to maintain homeostasis in the NAD⁺/NADH ratio, leading to the excess NADH produced in isobutanol production being consumed in wasteful reduction of other metabolic intermediates; e.g., by the production of lactate from pyruvate. Thus, the imbalance between NADH produced and NADPH consumed by the isobutanol pathway leads to a reduction in the molar yield of isobutanol produced from glucose in two ways: 1) unnecessary operation of metabolism to produce NADPH, and 2) wasteful reaction of metabolic intermediates to maintain NAD⁺/NADH homeostasis. The solution to this problem is to invent a KARI that is specific for NADH as its cofactor, so that both molecules of NADH produced in glycolysis are consumed in the synthesis of isobutanol from pyruvate.

Keto Acid Reductoisomerase (KARI) Enzymes

Acetohydroxy acid isomeroreductase or ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids and is a key enzyme in their biosynthesis. KARI is found in a variety of microorganisms and amino acid sequence comparisons across species have revealed that there are 2 types of this enzyme: a short form (class I) found in fungi and most bacteria, and a long form (class II) typical of plants.

Class I KARIs typically have between 330-340 amino acid residues. The long form KARI enzymes have about 490 amino acid residues. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. KARI is encoded by the ilvC gene and is an essential enzyme for growth of E. coli and other bacteria in a minimal medium. Typically KARI uses NADPH as cofactor and requires a divalent cation such as Mg⁺⁺ for its activity. In addition to utilizing acetolactate in the valine pathway, KARI also converts acetohydroxybutanoate to dihydroxymethylpentanoate in the isoleucine production pathway.

Class II KARIs generally consist of a 225-residue N-terminal domain and a 287-residue C-terminal domain. The N-terminal domain, which contains the NADPH-binding site, has an α/β structure and resembles domains found in other pyridine nucleotide-dependent oxidoreductases. The C-terminal domain consists almost entirely of α-helices and is of a previously unknown topology.

The crystal structure of the E. coli KARI enzyme at 2.6 Å resolution has been solved (Tyagi, et al., Protein Sci., 14: 3089-3100, 2005). This enzyme consists of two domains, one with mixed α/β structure which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly α-helical and shows strong evidence of internal duplication. Comparison of the active sites of KARI of E. coli, Pseudomonas aeruginosa, and spinach showed that most residues in the active site of the enzyme occupy conserved positions. While the E. coli KARI was crystallized as a tetramer, which is probably the likely biologically active unit, the P. aeruginosa KARI (Ahn, et al., J. Mol. Biol., 328: 505-515, 2003) formed a dodecamer, and the enzyme from spinach formed a dimer. Known KARIs are slow enzymes with a reported turnover number (k_(cat)) of 2 s⁻¹ (Aulabaugh et al.; Biochemistry, 29: 2824-2830, 1990) or 0.12 s⁻¹ (Rane et al., Arch. Biochem. Biophys. 338: 83-89, 1997) for acetolactate. Studies have shown that genetic control of isoleucine-valine biosynthesis in E. coli is different than that in Ps. aeruginosa (Marinus, et al., Genetics, 63: 547-56, 1969).

Identification of Amino Acid Target Sites for Cofactor Switching

It was reported that phosphate p2′ oxygen atoms of NADPH form hydrogen bonds with side chains of Arg162, Ser165 and Ser167 of spinach KARI (Biou V., et al. The EMBO Journal, 16: 3405-3415, 1997). Multiple sequence alignments were performed, using vector NTI (Invitrogen Corp. Carlsbad, Calif.), with KARI enzymes from spinach, Pseudomonas aeruginosa (PAO-KARI) and Pseudomonas fluorescens (PF5-KARI). The NADPH binding sites are shown in FIG. 2A. The amino acids, argenine, threonine and serine appear to play similar roles in forming hydrogen bonds with phosphate p2′ oxygen atoms of NADPH in KARI enzymes. Studies by Ahn et al., (J. Mol. Biol., 328: 505-515, 2003) had identified three NADPH phosphate binding sites (Arg47, Ser50 and Thr52) for Pseudomonas aeruginosa (PAO-KARI) following comparing its structure with that of the spinach KARI. Hypothesizing that these three NADPH phosphate binding sites of the three KARI enzymes used in the disclosure were conserved, Arg47, Ser50 and Thr52 of PF5-KARI were targeted as the phosphate binding sites for this enzyme. This hypothesis was further confirmed through homology modeling.

Multiple sequence alignment among PF5-ilvC and several other KARI enzymes with promiscuous nucleotide specificity was also performed. As shown in FIG. 2B, the amino acids of glycine (G50) and tryptophan (W53), in other KARI enzymes in FIG. 2B, always appear together as a pair in the sequences of those enzymes. It was therefore assumed that the tryptophan 53 bulky residue was important in determining nucleotide specificity and by reducing the size of nucleotide binding pocket one could favor binding of the smaller nucleotide, NADH. Position 53 of PF5-ilvC was therefore chosen as a target for mutagenesis.

Several site-saturation gene libraries were prepared containing genes encoding KARI enzymes by commercially available kits for the generation of mutants. Clones from each library were screened for improved KARI activity using the NADH consumption assay described herein. Screening resulted in the identification of a number of genes having mutations that can be correlated to KARI activity. The location of the mutations were identified using the amino acid sequence of the Pseudomonas fluorescens PF5 ilvC protein (SEQ ID NO:17). Mutants with improved KARI activity had mutations at one or more positions at amino acids: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, 165, and 170. More specifically desirable mutations included the following substitutions:

-   -   a) the residue at position 47 has an amino acid substitution         selected from the group consisting of A, C, D, F, G, I, L, N, P,         and Y;     -   b) the residue at position 50 has an amino acid substitution         selected from the group consisting of A, C, D, E, F, G, M, N, V,         W;     -   c) the residue at position 52 has an amino acid substitution         selected from the group consisting of A, C, D, G, H, N, S;     -   d) the residue at position 53 has an amino acid substitution         selected from the group consisting of A, H, I, W;

In another embodiment, additional mutagenesis, using error prone PCR, performed on the mutants listed above identified suitable mutation positions as: 156, 165, 61, 170, 115 and 24. More specifically the desirable mutants with lower K_(M) for NADH contained the following substitutions:

-   -   e) the residue at position 156 has an amino acid substitution of         V;     -   f) the residue at position 165 has an amino acid substitution of         M;     -   g) the residue at position 61 has an amino acid substitution of         F;     -   h) the residue at position 170 has an amino acid substitution of         A;     -   i) the residue at position 24 has an amino acid substitution of         F; and     -   j) the residue at position 115 has an amino acid substitution of         L.

In another embodiment, multiple sequence alignment of Pseudomonas fluorescens PF5-ilvC and Bacillus cereus ilvC1 and livC2 and spinach KARI was performed which allowed identification of positions 24, 33, 47, 50, 52, 53, 61, 80, 156 and 170 for further mutagenesis. More specifically mutants with much lower K_(M) for NADH were obtained. These mutations are also based on the Pseudomonas fluorescens, KARI enzyme (SEQ ID NO:17) as a reference sequence wherein the reference sequence comprises at least one amino acid substitution selected from the group consisting of:

-   -   k) the residue at position 24 has an amino acid substitution of         phenylalanine;     -   l) the residue at position 50 has an amino acid substitution of         alanine;     -   m) the residue at position 52 has an amino acid substitution of         aspartic acid;     -   n) the residue at position 53 has an amino acid substitution of         alanine;     -   o) the residue at position 61 has an amino acid substitution of         phenylalanine;     -   p) the residue at position 156 has an amino acid substitution of         valine;     -   q) the residue at position 33 has an amino acid substitution of         leucine;     -   r) the residue at position 47 has an amino acid substitution of         tyrosine;     -   s) the residue at position 80 has an amino acid substitution of         isoleucine;     -   and     -   t) the residue at position 170 has an amino acid substitution of         alanine.

The present invention includes a mutant polypeptide having KARI activity, said polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 and 28.

A consensus sequence for the mutant ilvC was generated from the multiple sequence alignment and is provided as SEQ ID NO: 29 which represents all experimentally verified mutations of the KARI enzyme based on the amino acid sequence of the KARI enzyme isolated from Pseudomonas fluorescens, (SEQ ID NO:17)

Additionally the present invention describes mutation positions identified using a profile Hidden Markov Model (HMM) built based on sequences of 25 functionally verified Class I and Class II KARI enzymes. Profile HMM identified mutation positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 (the numbering is based on the sequences of Pseudomonas fluorescens PF5 KARI). Thus, it will be appreciated by the skilled person that mutations at these positions, as well as those discussed above that have been experimentally verified will also give rise to KARI enzymes having the ability to bind NADH.

Furthermore, applicants have discovered that the ketol-acid reductoisomerase enzyme has two functionally related domains: one domain affecting nucleotide specificity and the other domain impacting the K_(M) for the cofactor (FIGS. 11 and 12). To examine whether this characteristic could be exploited to engineer the desired KARI mutants (i.e., mutants with high NADH activity (K_(M)<20 μM) and substantially decreased NADPH activity (K_(M)>100 μM)), two libraries were created.

One library was a four-site saturation library targeting the NADH or NADPH binding positions, i.e., amino acids at positions 47, 50, 52 and 53 (FIG. 11). To build this library, mutants which possessed both NADH and NADPH activities and K_(M)˜10-20 μM for NADH, were selected from a group consisting of SEQ ID NOs: 28, 67, 68, 69, 70 and 84, as templates. Further saturation mutagenesis generated new mutants (i.e., mutants with SEQ ID NOs: 75-78) that possessed mainly NADH activity with very low NADPH activity.

The desirable mutants with higher NADH activity, following site saturation mutagenesis, comprised the following substitutions:

-   -   u) the residue at position 24 has an amino acid substitution of         phenylalanine;     -   v) the residue at position 50 has an amino acid substitution of         aspartic acid or valine or isoleucine or phenylalanine;     -   w) the residue at position 52 has an amino acid substitution of         tyrosine or aspartic acid;     -   x) the residue at position 53 has an amino acid substitution of         tyrosine or glycine, or argenine, or alanine;     -   y) the residue at position 61 has an amino acid substitution of         phenylalanine;     -   z) the residue at position 156 has an amino acid substitution of         valine;     -   aa) the residue at position 33 has an amino acid substitution of         leucine;     -   bb) the residue at position 47 has an amino acid substitution of         histidine, or proline, or threonine, or glutamic acid; and     -   cc) the residue at position 80 has an amino acid substitution of         isoleucine.

The K_(M) for NADH in the above mutants was still slightly high (e.g., JB1C6, SEQ ID NO: 74, has K_(M) of 22 μM for NADH). To further improve the NADH K_(M) of the mutant KARIs, a “domain swapping library”, which combined the nucleotide switching mutations and mutations with improved K_(M) for NADH, was created (FIG. 12). More specifically, the beneficial mutations at positions 47, 50, 52 and 53 obtained in the site saturation experiment (see Tables 3 and 4), were transferred into mutants that possessed K_(M)˜4-40 μM for NADH (SEQ ID NOs:24-28 and 67-70 and 84, see Tables 6 and 7). The resultant new mutants accepted NADH as cofactor with very low K_(M)˜10 μM and greatly reduced NADPH activity. Examples of these mutants include: JEA1 (SEQ ID NO: 79), JEG2 (SEQ ID NO: 80), JEG4 (SEQ ID NO: 81), JEA7 (SEQ ID NO: 82) and JED1 (SEQ ID NO: 83).

Following domain swapping experiments, the mutants that possessed very low K_(M) for NADH had the following substitutions:

-   -   dd) the residue at position 24 has an amino acid substitution of         phenylalanine;     -   ee) the residue at position 50 has an amino acid substitution of         alanine, asparagine, or phenylalanine;     -   ff) the residue at position 52 has an amino acid substitution of         aspartic acid;     -   gg) the residue at position 53 has an amino acid substitution of         alanine;     -   hh) the residue at position 61 has an amino acid substitution of         phenylalanine;     -   ii) the residue at position 156 has an amino acid substitution         of valine;     -   jj) the residue at position 33 has an amino acid substitution of         leucine;     -   kk) the residue at position 47 has an amino acid substitution of         asparagine, proline; and phenylalanine;     -   ll) the residue at position 80 has an amino acid substitution of         isoleucine.

In one embodiment the present method includes a mutant polypeptide having KARI activity, said polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 24,-28, 67-70, and 75-98,

In another embodiment the method provides an NADH utilizing KARI mutant with a K_(M) for NADH <15 μM.

In a preferred embodiment, the mutant KARI JEA1 (SEQ ID NO: 79) has the following substitutions:

Y24F/C33L/R47P/S50F/T52D/L61F/T80I/A156V

In another preferred embodiment, the mutant KARI JEG2 (SEQ ID NO: 80) has the following substitutions:

(Y24F/C33L/R47F/S50A/T52D/V53A/L61F/T80I/A156V)

In another preferred embodiment, the mutant KARI JEG4 (SEQ ID NO: 81), has the following substitutions:

(Y24F/C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)

In another preferred embodiment, the mutant KARI JEA7 (SEQ ID NO: 82), has the following substitutions:

(Y24F/C33L/R47P/S50N/T52D/V53A/L61F/T80I/A156V)

In another preferred embodiment, the mutant KARI JED1 (SEQ ID NO: 83) has the following substitutions:

(C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)

In another embodiment the method provides an NADH accepting KARI mutant wherein the ratio of NADH/NADPH activity is greater than one. A consensus sequence for the mutant ilvC was generated from the multiple sequence alignment and is provided as SEQ ID NO: 29 which represents all experimentally verified mutations of the KARI enzyme based on the amino acid sequence of the KARI enzyme isolated from Pseudomonas fluorescens (SEQ ID NO:17).

The Host Strains for KARI Engineering

Two host strains, E. coli TOP10 from Invitrogen and E. coli Bw25113 (ΔilvC, an ilvC gene-knockout), were used for making constructs over-expressing the KARI enzyme in this disclosure. In the Bw25113 strain, the entire ilvC gene of the E. coli chromosome was replaced by a Kanamycin cassette using the Lambda red homology recombination technology described by Kirill et al., (Kirill A. Datsenko and Barry L. Wanner, Proc. Natl. Acad. Sci. USA, 97: 6640-6645, 2000).

Homology Modeling of PF5 KARI with Bound Substrates

The structure of PF5-KARI with bound NADPH, acetolactate and magnesium ions was built based on the crystal structure of P. aeruginosa PAO1-KARI (PDB ID 1NP3, Ahn H. J. et al., J. Mol. Biol., 328: 505-515, 2003) which has 92% amino acid sequence homology to PF5 KARI. PAO1-KARI structure is a homo-dodecamer and each dodecamer consists of six homo-dimers with extensive dimer interface. The active site of KARI is located in this dimer interface. The biological assembly is formed by six homo-dimers positioned on the edges of a tetrahedron resulting in a highly symmetrical dodecamer of 23 point group symmetry. For simplicity, only the dimeric unit (monomer A and monomer B) was built for the homology model of PF5-KARI in this study because the active site is in the homo-dimer interface.

The model of PF5-KARI dimer was built based on the coordinates of monomer A and monomer B of PAO1-KARI and sequence of PF5-KARI using DeepView/Swiss PDB viewer (Guex, N. and Peitsch, M. C., Electrophoresis, 18: 2714-2723, 1997). This model was then imported to program O (Jones, T. A. et al, Acta Crystallogr. A 47: 110-119, 1991) on a Silicon Graphics system for further modification.

The structure of PAO1-KARI has no NADPH, substrate or inhibitor or magnesium in the active site. Therefore, the spinach KARI structure (PDB ID 1yve, Biou V. et al., The EMBO Journal, 16: 3405-3415, 1997.), which has magnesium ions, NADPH and inhibitor (N-Hydroxy-N-isopropyloxamate) in the acetolacate binding site, was used to model these molecules in the active site. The plant KARI has very little sequence homology to either PF5- or PAO1 KARI (<20% amino acid identity), however the structures in the active site region of these two KARI enzymes are very similar. To overlay the active site of these two KARI structures, commands LSQ_ext, LSQ_improve, LSQ_mol in the program O were used to line up the active site of monomer A of spinach KARI to the monomer A of PF5 KARI model. The coordinates of NADPH, two magnesium ions and the inhibitor bound in the active site of spinach KARI were extracted and incorporated to molecule A of PF5 KARI. A set of the coordinates of these molecules were generated for monomer B of PF5 KARI by applying the transformation operator from monomer A to monomer B calculated by the program.

Because there is no NADPH in the active site of PAO1 KARI crystal structure, the structures of the phosphate binding loop region in the NADPH binding site (residues 44-45 in PAO1 KARI, 157-170 in spinach KARI) are very different between the two. To model the NADPH bound form, the model of the PF5-KARI phosphate binding loop (44-55) was replaced by that of 1yve (157-170). Any discrepancy of side chains between these two was converted to those in the PF5-KARI sequence using the mutate_replace command in program O, and the conformations of the replaced side-chains were manually adjusted. The entire NADPH/Mg/inhibitor bound dimeric PF5-KARI model went through one round of energy minimization using program CNX (ACCELRYS San Diego Calif., Burnger, A. T. and Warren, G. L., Acta Crystallogr., D 54: 905-921, 1998) after which the inhibitor was replaced by the substrate, acetolactate (AL), in the model. The conformation of AL was manually adjusted to favor hydride transfer of C4 of the nicotinamine of NADPH and the substrate. No further energy minimization was performed on this model (coordinates of the model created for this study are attached in a separate word file). The residues in the phosphate binding loop and their interactions with NADPH are illustrated in FIG. 3.

Application of a “Profile Hidden Markov Model” for Identification of Residue Positions Involved in Cofactor Switching in KARI Enzymes

Applicants have developed a method for identifying KARI enzymes and the residue positions that are involved in cofactor switching from NADPH to NADH. To structurally characterize KARI enzymes, a Profile Hidden Markov Model (HMM) was prepared as described in Example 5 using amino acid sequences of 25 KARI proteins with experimentally verified function as outlined in Table 6. These KARIs were from [Pseudomonas fluorescens Pf-5 (SEQ ID NO: 17), Sulfolobus solfataricus P2 (SEQ ID NO: 13), Pyrobaculum aerophilum str. IM2 (SEQ ID NO: 14), Natronomonas pharaonis DSM 2160 (SEQ ID NO: 30), Bacillus subtilis subsp. subtilis str. 168 (SEQ ID NO: 31), Corynebacterium glutamicum ATCC 13032 (SEQ ID NO: 32), Phaeospririlum molischianum (SEQ ID NO: 33), Ralstonia solanacearum GMI1000 (SEQ ID NO: 15), Zymomonas mobilis subsp. mobilis ZM4 (SEQ ID NO: 34), Alkalilimnicola ehrlichei MLHE-1 (SEQ ID NO: 35), Campylobacter lari RM2100 (SEQ ID NO: 36), Marinobacter aquaeolei VT8 (SEQ ID NO: 37), Psychrobacter arcticus 273-4 (SEQ ID NO: 38), Hahella chejuensis KCTC 2396 (SEQ ID NO: 39), Thiobacillus denitrificans ATCC 25259 (SEQ ID NO: 40), Azotobacter vinelandii AvOP (SEQ ID NO: 41), Pseudomonas syringae pv. syringae B728a (SEQ ID NO: 42), Pseudomonas syringae pv. tomato str. DC3000 (SEQ ID NO: 43), Pseudomonas putida KT2440 (Protein SEQ ID NO: 44), Pseudomonas entomophila L48 (SEQ ID NO: 45), Pseudomonas mendocina ymp (SEQ ID NO: 46), Pseudomonas aeruginosa PAO1 (SEQ ID NO: 16), Bacillus cereus ATCC 10987 (SEQ ID NO: 47), Bacillus cereus ATCC 10987 (SEQ ID NO: 48), and Spinacia oleracea (SEQ ID NO: 18).

In addition using methods disclosed in this application, sequences of Class II KARI enzymes such as E. coli (SEQ ID NO: 63—GenBank Accession Number P05793), marine gamma Proteobacterium HTCC2207 (SEQ ID NO: 64—GenBank Accession Number ZP_(—)01224863.1), Desulfuromonas acetoxidans (SEQ ID NO: 65—GenBank Accession Number ZP_(—)01313517.1) and Pisum sativum (pea) (SEQ ID NO: 66—GenBank Accession Number O82043) could be mentioned.

This Profile HMM for KARIs may be used to identify any KARI related proteins. Any protein that matches the Profile HMM with an E value of <10⁻³ using hmmsearch program in the HMMER package is expected to be a functional KARI, which can be either a Class I and Class II KARI. Sequences matching the Profile HMM given herein are then analyzed for the location of the 12 positions in Pseudomonas fluorescens Pf-5 that switches the cofactor from NADPH to NADH. The eleven nodes, as defined in the section of Profile HMM building, in the profile HMM representing the columns in the alignment which correspond to the eleven co-factor switching positions in Pseudomonas fluorescens Pf-5 KARI are identified as node 24, 33, 47, 50, 52, 53, 61, 80, 115, 156 and 170. The lines corresponding to these nodes in the model file are identified in Table 9. One skilled in the art will readily be able to identify these 12 positions in the amino acid sequence of a KARI protein from the alignment of the sequence to the profile HMM using hmm search program in HMMER package.

The KARI enzymes identified by this method, include both Class I and Class II KARI enzymes from either microbial or plant natural sources. Any KARI identified by this method may be used for heterologous expression in microbial cells.

For example each of the KARI encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1) methods of nucleic acid hybridization; 2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR) (Mullis et al., U.S. Pat. No. 4,683,202); ligase chain reaction (LCR) (Tabor, S. et al., Proc. Acad. Sci. USA 82:1074, 1985); or strand displacement amplification (SDA) (Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89: 392, 1992); and 3) methods of library construction and screening by complementation.

Although the sequence homology between Class I and Class II KARI enzymes is low, the three dimensional structure of both Classes of the enzymes, particularly around the active site and nucleotide binding domains is highly conserved (Tygai, R., et al., Protein Science, 34: 399-408, 2001). The key amino acid residues that make up the substrate binding pocket are highly conserved between these two Classes even though they may not align well in a simple sequence comparison. It can therefore be concluded that the residues affecting cofactor specificity identified in Class I KARI (e.g., positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 of PF5 KARI) can be extended to Class II KARI enzymes.

Isobutanol Biosynthetic Pathways

Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Parnas (EMP) pathway, the Entner and Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP) as the central, metabolic routes to provide energy and cellular precursors for growth and maintenance. These pathways have in common the intermediate glyceraldehyde-3-phosphate and, ultimately, pyruvate is formed directly or in combination with the EMP pathway. Subsequently, pyruvate is transformed to acetyl-cofactor A (acetyl-CoA) via a variety of means. Acetyl-CoA serves as a key intermediate, for example, in generating fatty acids, amino acids and secondary metabolites. The combined reactions of sugar conversion to pyruvate produce energy (e.g., adenosine-5′-triphosphate, ATP) and reducing equivalents (e.g., reduced nicotinamide adenine dinucleotide, NADH, and reduced nicotinamide adenine dinucleotide phosphate, NADPH). NADH and NADPH must be recycled to their oxidized forms (NAD⁺ and NADP⁺, respectively). In the presence of inorganic electron acceptors (e.g. O₂, NO₃ ⁻ and SO₄ ²⁻), the reducing equivalents may be used to augment the energy pool; alternatively, a reduced carbon byproduct may be formed.

There are four potential pathways for production of isobutanol from carbohydrate sources with recombinant microorganisms as shown in FIG. 1. All potential pathways for conversion of carbohydrates to isobutanol have been described in the commonly owned U.S. patent application Ser. No. 11/586,315, which is incorporated herein by reference.

The preferred pathway for conversion of pyruvate to isobutanol consists of enzymatic steps “a”, “b”, “c”, “d”, and “e” (FIGS. 1A and 1B) and includes the following substrate to product conversions:

-   -   a) pyruvate to acetolactate, as catalyzed for example by         acetolactate synthase,     -   b) (S)-acetolactate to 2,3-dihydroxyisovalerate, as catalyzed         for example by acetohydroxy acid isomeroreductase,     -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed         for example by acetohydroxy acid dehydratase,     -   d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for         example by a branched-chain keto acid decarboxylase, and     -   e) isobutyraldehyde to isobutanol, as catalyzed for example by,         a branched-chain alcohol dehydrogenase.

This pathway combines enzymes involved in well-characterized pathways for valine biosynthesis (pyruvate to α-ketoisovalerate) and valine catabolism (α-ketoisovalerate to isobutanol). Since many valine biosynthetic enzymes also catalyze analogous reactions in the isoleucine biosynthetic pathway, substrate specificity is a major consideration in selecting the gene sources. For this reason, the primary genes of interest for the acetolactate synthase enzyme are those from Bacillus (alsS) and Klebsiella (budB). These particular acetolactate synthases are known to participate in butanediol fermentation in these microorganisms and show increased affinity for pyruvate over ketobutyrate (Gollop et al., J. Bacteriol., 172: 3444-3449, 1990); and (Holtzclaw et al., J. Bacteriol., 121: 917-922, 1975). The second and third pathway steps are catalyzed by acetohydroxy acid reductoisomerase and dehydratase, respectively. These enzymes have been characterized from a number of sources, such as for example, E. coli (Chunduru et al., Biochemistry, 28: 486-493, 1989); and (Flint et al., J. Biol. Chem., 268: 14732-14742, 1993). The final two steps of the preferred isobutanol pathway are known to occur in yeast, which can use valine as a nitrogen source and, in the process, secrete isobutanol. α-Ketoiso-valerate can be converted to isobutyraldehyde by a number of keto acid decarboxylase enzymes, such as for example pyruvate decarboxylase. To prevent misdirection of pyruvate away from isobutanol production, a decarboxylase with decreased affinity for pyruvate is desired. So far, there are two such enzymes known in the art (Smit et al., Appl. Environ. Microbiol., 71: 303-311, 2005); and (de la Plaza et al., FEMS Microbiol. Lett., 238: 367-374, 2004). Both enzymes are from strains of Lactococcus lactis and have a 50-200-fold preference for ketoisovalerate over pyruvate. Finally, a number of aldehyde reductases have been identified in yeast, many with overlapping substrate specificity. Those known to prefer branched-chain substrates over acetaldehyde include, but are not limited to, alcohol dehydrogenase VI (ADH6) and Ypr1p (Larroy et al., Biochem. J., 361: 163-172, 2002); and (Ford et al., Yeast, 19: 1087-1096, 2002), both of which use NADPH as electron donor. An NADPH-dependent reductase, YqhD, active with branched-chain substrates has also been recently identified in E. coli (Sulzenbacher et al., J. Mol. Biol., 342: 489-502, 2004).

Two of the other potential pathways for isobutanol production also contain the initial three steps of “a”, “b” and “c” (FIG. 1A). One pathway consists of enzymatic steps “a”, “b”, “c”, “f”, “g”, “e” (FIGS. 1A and 1B). Step “f” containing a “branched-chain keto acid dehydrogenase (EC1.2.4.4). Step “g” containing an “acylating aldehyde dehydrogenase” (EC1.2.1.10) and 1.2.1.57 in addition to step “e” containing the “branched chain alcohol dehydrogenase”. The other potential pathway consists of steps “a”, “b”, “c”, “h”, “i”, “j”, “e” (FIGS. 1A and 1B). The term “transaminase” (step “h”) EC numbers 2.6.1.42 and 2.6.1.66. Step “h” consists of either a “valine dehydrogenase” (EC1.4.1.8 and EC1.4.1.9) or step “i”, a “valine decarboxylase” with an EC number 4.1.1.14. Finally step “j” will use an “omega transaminase” (EC2.6.1.18) to generate isobutyraldehyde which will be reduced by step “e” to produce isobutanol. All potential pathways for conversion of pyruvate to isobutanol are depicted in FIGS. 1A and 1B.

Additionally, a number of microorganisms are known to produce butyrate and/or butanol via a butyryl-CoA intermediate (Dürre, et al., FEMS Microbiol. Rev., 17: 251-262, 1995); and (Abbad-Andaloussi et al., Microbiology, 142: 1149-1158, 1996). Therefore isobutanol production in these microorganisms will take place using steps “k”, “g” and “e” shown in FIG. 1B. Step “k” will use an “isobutyryl-CoA mutase” (EC5.4.99.13). The nest step will involve using the “acylating aldehyde dehydrogenase” (EC 1.2.1.10 and EC1.2.1.57) to produce isobutyraldehyde followed by enzymatic step “e” to produce isobutanol. All these pathways are fully described in the commonly owned patent application Ser. No. 11/586,315, herein incorporated by reference.

Thus, in providing multiple recombinant pathways from pyruvate to isobutanol, there exist a number of choices to fulfill the individual conversion steps, and the person of skill in the art will be able to use publicly available sequences to construct the relevant pathways.

Microbial Hosts for Isobutanol Production

Microbial hosts for isobutanol production may be selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microbial host used for isobutanol production should be tolerant to isobutanol so that the yield is not limited by butanol toxicity. Microbes that are metabolically active at high titer levels of isobutanol are not well known in the art. Although butanol-tolerant mutants have been isolated from solventogenic Clostridia, little information is available concerning the butanol tolerance of other potentially useful bacterial strains. Most of the studies on the comparison of alcohol tolerance in bacteria suggest that butanol is more toxic than ethanol (de Cavalho, et al., Microsc. Res. Tech., 64: 215-22, 2004) and (Kabelitz, et al., FEMS Microbiol. Lett., 220: 223-227, 2003, Tomas, et al., J. Bacteriol., 186: 2006-2018, 2004) report that the yield of 1-butanol during fermentation in Clostridium acetobutylicum may be limited by 1-butanol toxicity. The primary effect of 1-butanol on Clostridium acetobutylicum is disruption of membrane functions (Hermann et al., Appl. Environ. Microbiol., 50: 1238-1243, 1985).

The microbial hosts selected for the production of isobutanol should be tolerant to isobutanol and should be able to convert carbohydrates to isobutanol. The criteria for selection of suitable microbial hosts include the following: intrinsic tolerance to isobutanol, high rate of glucose utilization, availability of genetic tools for gene manipulation, and the ability to generate stable chromosomal alterations.

Suitable host strains with a tolerance for isobutanol may be identified by screening based on the intrinsic tolerance of the strain. The intrinsic tolerance of microbes to isobutanol may be measured by determining the concentration of isobutanol that is responsible for 50% inhibition of the growth rate (IC₅₀) when grown in a minimal medium. The IC₅₀ values may be determined using methods known in the art. For example, the microbes of interest may be grown in the presence of various amounts of isobutanol and the growth rate monitored by measuring the optical density at 600 nanometers. The doubling time may be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of isobutanol that produces 50% inhibition of growth may be determined from a graph of the percent inhibition of growth versus the isobutanol concentration. Preferably, the host strain should have an IC₅₀ for isobutanol of greater than about 0.5%.

The microbial host for isobutanol production should also utilize glucose at a high rate. Most microbes are capable of metabolizing carbohydrates. However, certain environmental microbes cannot metabolize carbohydrates to high efficiency, and therefore would not be suitable hosts.

The ability to genetically modify the host is essential for the production of any recombinant microorganism. The mode of gene transfer technology may be by electroporation, conjugation, transduction or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host microorganisms based on the nature of antibiotic resistance markers that can function in that host.

The microbial host also has to be manipulated in order to inactivate competing pathways for carbon flow by deleting various genes. This requires the availability of either transposons to direct inactivation or chromosomal integration vectors. Additionally, the production host should be amenable to chemical mutagenesis so that mutations to improve intrinsic isobutanol tolerance may be obtained.

Based on the criteria described above, suitable microbial hosts for the production of isobutanol include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae.

Construction of Production Host

Recombinant microorganisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a fermentable carbon substrate to isobutanol may be constructed using techniques well known in the art. In the present invention, genes encoding the enzymes of one of the isobutanol biosynthetic pathways of the invention, for example, acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain α-keto acid decarboxylase, and branched-chain alcohol dehydrogenase, may be isolated from various sources, as described above.

Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available. Some tools for codon optimization are available based on the GC content of the host microorganism.

Once the relevant pathway genes are identified and isolated they may be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.). Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans.

Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.

Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors-pRK437, pRK442, and pRK442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid, 50: 74-79, 2003). Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for the heterologous gene expression in Gram-negative bacteria.

Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad-host-range replicon pWV101 has been modified to construct a plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria (Maguin et al., J. Bacteriol., 174: 5633-5638, 1992). Additionally, in vitro transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE®.

The expression of an isobutanol biosynthetic pathway in various preferred microbial hosts is described in more detail below.

Expression of an Isobutanol Biosynthetic Pathway in E. coli

Vectors or cassettes useful for the transformation of E. coli are common and commercially available from the companies listed above. For example, the genes of an isobutanol biosynthetic pathway may be isolated from various sources, cloned into a modified pUC19 vector and transformed into E. coli NM522.

Expression of an Isobutanol Biosynthetic Pathway in Rhodococcus erythropolis

A series of E. coli-Rhodococcus shuttle vectors are available for expression in R. erythropolis, including, but not limited to, pRhBR17 and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol., 62: 61-68, 2003). Additionally, a series of promoters are available for heterologous gene expression in R. erythropolis (Nakashima et al., Appl. Environ. Microbiol., 70: 5557-5568, 2004 and Tao et al., Appl. Microbiol. Biotechnol., 68: 346-354, 2005). Targeted gene disruption of chromosomal genes in R. erythropolis may be created using the method described by Tao et al., supra, and Brans et al. (Appl. Environ. Microbiol., 66: 2029-2036, 2000).

The heterologous genes required for the production of isobutanol, as described above, may be cloned initially in pDA71 or pRhBR71 and transformed into E. coli. The vectors may then be transformed into R. erythropolis by electroporation, as described by Kostichka et al., supra. The recombinants may be grown in synthetic medium containing glucose and the production of isobutanol can be followed using methods known in the art.

Expression of an Isobutanol Biosynthetic Pathway in B. subtilis

Methods for gene expression and creation of mutations in B. subtilis are also well known in the art. For example, the genes of an isobutanol biosynthetic pathway may be isolated from various sources, cloned into a modified pUC19 vector and transformed into Bacillus subtilis BE1010. Additionally, the five genes of an isobutanol biosynthetic pathway can be split into two operons for expression. The three genes of the pathway (bubB, ilvD, and kivD) can be integrated into the chromosome of Bacillus subtilis BE1010 (Payne, et al., J. Bacteriol., 173, 2278-2282, 1991). The remaining two genes (ilvC and bdhB) can be cloned into an expression vector and transformed into the Bacillus strain carrying the integrated isobutanol genes

Expression of an Isobutanol Biosynthetic Pathway in B. licheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtilis may be used to transform B. licheniformis by either protoplast transformation or electroporation. The genes required for the production of isobutanol may be cloned in plasmids pBE20 or pBE60 derivatives (Nagarajan et al., Gene, 114: 121-126, 1992). Methods to transform B. licheniformis are known in the art (Fleming et al. Appl. Environ. Microbiol., 61: 3775-3780, 1995). The plasmids constructed for expression in B. subtilis may be transformed into B. licheniformis to produce a recombinant microbial host that produces isobutanol.

Expression of an Isobutanol Biosynthetic Pathway in Paenibacillus macerans

Plasmids may be constructed as described above for expression in B. subtilis and used to transform Paenibacillus macerans by protoplast transformation to produce a recombinant microbial host that produces isobutanol.

Expression of the Isobutanol Biosynthetic Pathway in Alcaliqenes (Ralstonia) eutrophus

Methods for gene expression and creation of mutations in Alcaligenes eutrophus are known in the art (Taghavi et al., Appl. Environ. Microbiol., 60: 3585-3591, 1994). The genes for an isobutanol biosynthetic pathway may be cloned in any of the broad host range vectors described above, and electroporated to generate recombinants that produce isobutanol. The poly(hydroxybutyrate) pathway in Alcaligenes has been described in detail, a variety of genetic techniques to modify the Alcaligenes eutrophus genome is known, and those tools can be applied for engineering an isobutanol biosynthetic pathway.

Expression of an Isobutanol Biosynthetic Pathway in Pseudomonas putida

Methods for gene expression in Pseudomonas putida are known in the art (see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which is incorporated herein by reference). The butanol pathway genes may be inserted into pPCU18 and this ligated DNA may be electroporated into electrocompetent Pseudomonas putida DOT-T1 C5aAR1 cells to generate recombinants that produce isobutanol.

Expression of an Isobutanol Biosynthetic Pathway in Saccharomyces cerevisiae

Methods for gene expression in Saccharomyces cerevisiae are known in the art (e.g., Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology, Part A, 2004, Christine Guthrie and Gerald R. Fink, eds., Elsevier Academic Press, San Diego, Calif.). Expression of genes in yeast typically requires a promoter, followed by the gene of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes encoding an isobutanol biosynthetic pathway, including, but not limited to constitutive promoters FBA, GPD, ADH1, and GPM, and the inducible promoters GAL1, GAL10, and CUP1. Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1. For example, suitable promoters, transcriptional terminators, and the genes of an isobutanol biosynthetic pathway may be cloned into E. coli-yeast shuttle vectors.

Expression of an Isobutanol Biosynthetic Pathway in Lactobacillus plantarum

The Lactobacillus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus may be used for lactobacillus. Non-limiting examples of suitable vectors include pAMβ1 and derivatives thereof (Renault et al., Gene 183:175-182, 1996); and (O'Sullivan et al., Gene, 137: 227-231, 1993); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al., Appl. Environ. Microbiol., 62: 1481-1486, 1996); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol., 184: 5800-5804, 2002); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol., 63: 4581-4584, 1997); pAM401 (Fujimoto et al., Appl. Environ. Microbiol., 67: 1262-1267, 2001); and pAT392 (Arthur et al., Antimicrob. Agents Chemother., 38: 1899-1903, 1994). Several plasmids from Lactobacillus plantarum have also been reported (van Kranenburg R, et al. Appl. Environ. Microbiol., 71: 1223-1230, 2005).

Expression of an Isobutanol Biosynthetic Pathway in Various Enterococcus Species (E. faecium, E. gallinarium, and E. faecalis)

The Enterococcus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Lactobacilli, Bacilli and Streptococci species may be used for Enterococcus species. Non-limiting examples of suitable vectors include pAMβ1 and derivatives thereof (Renault et al., Gene, 183: 175-182, 1996); and (O'Sullivan et al., Gene, 137: 227-231, 1993); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl. Environ. Microbiol., 62: 1481-1486, 1996); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol., 184: 5800-5804, 2002); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol., 63: 4581-4584, 1997); pAM401 (Fujimoto et al., Appl. Environ. Microbiol., 67: 1262-1267, 2001); and pAT392 (Arthur et al., Antimicrob. Agents Chemother., 38: 1899-1903, 1994). Expression vectors for E. faecalis using the nisA gene from Lactococcus may also be used (Eichenbaum et al., Appl. Environ. Microbiol., 64: 2763-2769, 1998). Additionally, vectors for gene replacement in the E. faecium chromosome may be used (Nallaapareddy et al., Appl. Environ. Microbiol., 72: 334-345, 2006).

Fermentation Media

Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates methylotrophic microorganisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. (eds): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol., 153: 485-489, 1990). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of microorganism.

Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for growth of the cultures and promotion of the enzymatic pathway necessary for isobutanol production.

Culture Conditions

Typically cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′,3′-monophosphate (cAMP), may also be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred for the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.

Industrial Batch and Continuous Fermentations

The present process employs a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired microorganism or microorganisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO₂. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund (Appl. Biochem. Biotechnol., 36: 227, 1992), herein incorporated by reference.

Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth may be altered continuously while the cell concentration, measured by medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol production.

Methods for Isobutanol Isolation from the Fermentation Medium

The biologically produced isobutanol may be isolated from the fermentation medium using methods known in the art for Acetone-butanol-ethanol (ABE) fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49: 639-648, 1998), and (Groot et al., Process. Biochem. 27: 61-75, 1992 and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation and isobutanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Materials and Methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C., 1994, or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.

The meaning of abbreviations used is as follows: “A” means Angstrom, “min” means minute(s), “h” means hour(s), “μl” means microliter(s), “ng/μl” means nano gram per microliter, “pmol/μl” means pico mole per microliter, “ml” means milliliter(s), “L” means liter(s), “g/L” mean gram per liter, “ng” means nano gram, “sec” means second(s), “ml/min” means milliliter per minute(s), “w/v” means weight per volume, “v/v” means volume per volume, “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” means micromole(s), g” means gram(s), “μg” means microgram(s), “mg” means milligram(s), “g” means the gravitation constant, “rpm” means revolutions per minute, “HPLC” means high performance liquid chromatography, “MS” means mass spectrometry, “HPLC/MS” means high performance liquid chromatography/mass spectrometry, “EDTA” means ethylendiamine-tetraacetic acid, “dNTP” means deoxynucleotide triphosphate, “° C.” means degrees Celsius, and “V” means voltage.

The oligonucleotide primers used in the following Examples have been described herein (see Table 1).

High Throughput Screening Assay of Gene Libraries

High throughput screening of the gene libraries of mutant KARI enzymes was performed as described herein: 10× freezing medium containing 554.4 g/L glycerol, 68 mM of (NH₄)₂SO₄, 4 mM MgSO₄, 17 mM sodium citrate, 132 mM KH₂PO₄, 36 mM K₂HPO₄ was prepared with molecular pure water and filter-sterilized. Freezing medium was prepared by diluting the 10× freezing medium with the LB medium. An aliquot (200 μl) of the freezing medium was used for each well of the 96-well archive plates (cat #3370, Corning Inc. Corning, N.Y.).

Clones from the LB agar plates were selected and inoculated into the 96-well archive plates containing the freezing medium and grown overnight at 37° C. without shaking. The archive plates were then stored at −80° C. E. coli strain Bw25113 transformed with pBAD-HisB (Invitrogen) was always used as the negative control. For libraries C, E, F and G, mutant T52D of (PF5-ilvC) was used as the positive control. The mutant T52D was a mutant of PF5-ilvC in which the threonine at position 52 was changed to aspartic acid. For library H, mutant C3B11 (R47F/S50A/T52D/v53W of PF5-ilvC) was used as the positive control.

Clones from archive plates were inoculated into the 96-deep well plates. Each well contained 3.0 μl of cells from thawed archive plates, 300 μl of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v) arabinose as the inducer. Cells were the grown overnight at 37° C. with 80% humidity while shaking (900 rpm), harvested by centrifugation (4000 rpm, 5 min at 25° C.). (Eppendorf centrifuge, Brinkmann Instruments, Inc. Westbury, N.Y.) and the cell pellet was stored at −20° C. for later analysis.

The assay substrate, (R,S)-acetolactate, was synthesized as described by Aulabaugh and Schloss (Aulabaugh and Schloss, Biochemistry, 29: 2824-2830, 1990): 1.0 g of 2-acetoxy-2-methyl-3-oxobutyric acid ethyl ester (Aldrich, Milwaukee, Wis.) was mixed with 10 ml NaOH (1.0 M) and stirred at room temperature. When the solution's pH became neutral, additional NaOH was slowly added until pH ˜8.0 was maintained. All other chemicals used in the assay were purchased from Sigma.

The enzymatic conversion of acetolactate to α,β-dihydroxyisovalerate by KARI was followed by measuring the disappearance of the cofactor, NADPH or NADH, from the reaction at 340 nm using a plate reader (Molecular Device, Sunnyvale, Calif.). The activity was calculated using the molar extinction coefficient of 6220 M⁻¹ cm⁻¹ for either NADPH or NADH. The stock solutions used were: K₂HPO₄ (0.2 M); KH₂PO₄ (0.2 M); EDTA (0.5 M); MgCl₂ (1.0 M); NADPH (2.0 mM); NADH (2.0 mM) and acetolactate (45 mM). The 100 ml reaction buffer mix stock containing: 4.8 ml K₂HPO₄, 0.2 ml KH₂PO₄, 4.0 ml MgCl₂, 0.1 ml EDTA and 90.9 ml water was prepared.

Frozen cell pellet in deep-well plates and BugBuster were warmed up at room temperature for 30 min at the same time. Each well of 96-well assay plates was filled with 120 μl of the reaction buffer and 20 μl of NADH (2.0 mM), 150 μl of BugBuster was added to each well after 30 min warm-up and cells were suspended using Genmate (Tecan Systems Inc. San Jose, Calif.) by pipetting the cell suspension up and down (×5). The plates were incubated at room temperature for 20 min and then heated at 60° C. for 10 min. The cell debris and protein precipitates were removed by centrifugation at 4,000 rpm for 5 min at 25° C. An aliquot (50 μl) of the supernatant was transferred into each well of 96-well assay plates, the solution was mixed and the bubbles were removed by centrifugation at 4,000 rpm at 25° C. for 1 min. Absorbance at 340 nm was recorded as background, 20 μl of acetolactate (4.5 mM, diluted with the reaction buffer) was added to each well and mixed with shaking by the plate reader. Absorbance at 340 nm was recoded at 0, and 60 minutes after substrate addition. The difference in absorbance (before and after substrate addition) was used to determine the activity of the mutants. Mutants with higher KARI activity compared to the wild type were selected for re-screening.

About 5,000 clones were screened for library C and 360 top performers were selected for re-screen. About 92 clones were screened for library E and 16 top performers were selected for re-screening. About 92 clones were screened for library F and 8 top performers were selected for re-screening. About 92 clones were screened for library G and 20 top performers were selected for re-screening. About 8,000 clones were screened for library H and 62 top performers were selected for re-screening. The re-screening was described below as secondary assay.

Secondary Assay of Active Mutants

Cells containing pBad-ilvC and its mutants identified by high throughput screening were grown overnight, at 37° C., in 3.0 ml of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v) arabinose as the inducer while shaking at 250 rpm. The cells were then harvested by centrifugation at 18,000×g for 1 min at room temperature (Sigma micro-centrifuge model 1-15, Laurel, Md.). The cell pellets were re-suspended in 300 μl of BugBuster Master Mix (EMD Chemicals). The reaction mixture was first incubated at room temperature for 20 min and then heated at 60° C. for 10 min. The cell debris and protein precipitate were removed by centrifugation at 18,000×g for 5 min at room temperature.

The reaction buffer (120 μl) prepared as described above was mixed with either NADH or NADPH (20 μl) stock and cell extract (20 μl) in each well of a 96-well assay plate. The absorbance at 340 nm at 25° C. was recorded as background. Then 20 μl of acetolactate (4.5 mM, diluted with reaction buffer) was added each well and mixed with shaking by the plate reader. The absorbance at 340 nm at 0 min, 2 min and 5 min after adding acetolactate was recorded. The absorbance difference before and after adding substrate was used to determine the activity of the mutants. The mutants with high activity were selected for sequencing.

Five top performers from “Library C” were identified and sequenced (FIG. 4). The best performer was mutant R47F/S50A/T52D/V53W, which completely reversed the nucleotide specificity. The best performers from “Libraries E, F and G” were R47P, S50D and T52D respectively (FIG. 5). For “Library H”, 5 top performers were identified and sequenced (FIG. 6) and the best performer was R47P/S50G/T52D, which also completely reversed the nucleotide specificity. Enzymes containing activities higher than the background were considered positive.

KARI Enzyme Assay

KARI enzyme activity can be routinely measured by NADH or NADPH oxidation as described above, however to measure formation of the 2,3-dihydroxyisovalerate product directly, analysis of the reaction was performed using HPLC/MS.

Protein concentration of crude cell extract from Bugbuster lysed cells (as described above) was measured using the BioRad protein assay reagent (BioRad Laboratories, Inc., Hercules, Calif. 94547). A total of 0.5 micrograms of crude extract protein was added to a reaction buffer consisting of 100 mM HEPES-KOH, pH 7.5, 10 mM MgCl₂, 1 mM glucose-6-phosphate (Sigma-Aldrich), 0.2 Units of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Sigma-Aldrich), and various concentrations of NADH or NADPH, to a volume of 96 μL. The reaction was initiated by the addition of 4 μL of acetolactate to a final concentration of 4 mM and a final volume of 100 μL. After timed incubations at 30° C., typically between 2 and 15 min, the reaction was quenched by the addition of 10 μL of 0.5 M EDTA, pH 8.0 (Life Technologies, Grand Island, N.Y. 14072). To measure the K_(M) of NADH, the concentrations used were 0.03, 0.1, 0.3, 1, 3, and 10 mM.

To analyze for 2,3-dihydroxyisovalerate, the sample was diluted 10× with water, and 8.0 μl was injected into a Waters Acquity HPLC equipped with Waters SQD mass spectrometer (Waters Corporation, Milford, Mass.). The chromatography conditions were: flow rate (0.5 ml/min), on a Waters Acquity HSS T3 column (2.1 mm diameter, 100 mm length). Buffer A consisted of 0.1% (v/v) in water, Buffer B was 0.1% formic acid in acetonitrile. The sample was analyzed using 1% buffer B (in buffer A) for 1 min, followed by a linear gradient from 1% buffer B at 1 min to 75% buffer B at 1.5 min. The reaction product, 2,3-dihydroxyisovalerate, was detected by ionization at m/z=133, using the electrospray ionization devise at −30 V cone voltage. The amount of product 2,3-dihydroxyisovalerate was calculated by comparison to an authentic standard.

To calculate the K_(M) for NADH, the rate data for DHIV formation was plotted in Kaleidagraph (Synergy Software, Reading, Pa.) and fitted to the single substrate Michaelis-Menton equation, assuming saturating acetolactate concentration.

Example 1 Construction of Site-Saturation Gene Libraries to Identify Mutants Accepting NADH as Cofactor

Seven gene libraries were constructed (Table 2) using two steps: 1) synthesis of Megaprimers using commercially synthesized oligonucleotides described in Table 1; and 2) construction of mutated genes using the Megaprimers obtained in step 1. These primers were prepared using high fidelity pfu-ultra polymerase (Stratagene, La Jolla, Calif.) for one pair of primer containing one forward and one reverse primer. The templates for libraries C, E, F, G and H were the wild type of PF5_ilvc. The DNA templates for library N were those mutants having detectable NADH activity from library C while those for library O were those mutants having detectable NADH activity from library H. A 50 μl reaction mixture contained: 5.0 μl of 10× reaction buffer supplied with the pfu-ultra polymerase (Stratagene), 1.0 μl of 50 ng/μl template, 1.0 μl each of 10 pmol/μl forward and reverse primers, 1.0 μl of 40 mM dNTP mix (Promega, Madison, Wis.), 1.0 μl pfu-ultra DNA polymerase (Stratagene) and 39 μl water. The mixture was placed in a thin well 200 μl tube for the PCR reaction in a Mastercycler gradient equipment (Brinkmann Instruments, Inc. Westbury, N.Y.). The following conditions were used for the PCR reaction: The starting temperature was 95° C. for 30 sec followed by 30 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 54° C. for 1 min, and 70° C. for 2 min. At the completion of the temperature cycling, the samples were kept at 70° C. for 4 min more, and then held awaiting sample recovery at 4° C. The PCR product was cleaned up using a DNA cleaning kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer.

TABLE 2 Gene Libraries Library Targeted position(s) name Templates of Pf5_ilvC Primers used C PF5_ilvc 47, 50, 52 and 53 SEQ ID No: 1 and 2 E PF5_ilvc 47 SEQ ID No: 1 and 3 F PF5_ilvc 50 SEQ ID No: 1 and 4 G PF5_ilvc 52 SEQ ID No: 1 and 5 H PF5_ilvc 47, 50, and 52 SEQ ID No: 1 and 6 N Good mutants 53 SEQ ID NO: 20 and 21 from library C O Good mutants 53 SEQ ID NO: 20 and 21 from library H

The Megaprimers were then used to generate gene libraries using the QuickChange II XL site directed mutagenesis kit (Catalog #200524, Stratagene, La Jolla Calif.). A 50 μl reaction mixture contained: 5.0 μl of 10× reaction buffer, 1.0 μl of 50 ng/μl template, 42 μl Megaprimer, 1.0 μl of 40 mM dNTP mix, 1.0 μl pfu-ultra DNA polymerase. Except for the Megaprimer and the templates, all reagents used here were supplied with the kit indicated above. This reaction mixture was placed in a thin well 200 μl-capacity PCR tube and the following reactions were used for the PCR: The starting temperature was 95° C. for 30 sec followed by 25 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 1 min, and 68° C. for 6 min. At the completion of the temperature cycling, the samples were kept at 68° C. for 8 min more, and then held at 4° C. for later processing. Dpn I restriction enzyme (1.0 μl) (supplied with the kit above) was directly added to the finished reaction mixture, enzyme digestion was performed at 37° C. for 1 h and the PCR product was cleaned up using a DNA cleaning kit (Zymo Research). The cleaned PCR product (10 μl) contained mutated genes for a gene library.

The cleaned PCR product was transformed into an electrocompetent strain of E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, Calif.). The transformed clones were streaked on agar plates containing the LB medium and 100 μg/ml ampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Dozens of clones were randomly chosen for DNA sequencing to confirm the quality of the library.

TABLE 3 List of some mutants having NADH activity identified from saturation libraries Mutant Position 47 Position 50 Position 52 Position 53 SD2 R47Y S50A T52H V53W SB1 R47Y S50A T52G V53W SE1 R47A S50W T52G V53W SH2 R47N S50W T52N V53W SB2 R47I T52G V53W SG1 R47Y T52G V53W SB3 R47G S50W T52G V53W SE2 R47P S50E T52A V53W SD3 R47L S50W T52G V53W C2A6 R47I S50G T52D V53W C3E11 R47A S50M T52D V53W C3A7 R47Y S50A T52D V53W C3B11 R47F S50A T52D V53W C4A5 R47Y S50A T52S V53W C3B12 R47I T52D V53W C4H7 R47I T52S V53W C1D3 R47G S50M T52D V53W C4D12 R47C S50W T52G V53W C1G7 R47P S50G T52D V53W C2F6 R47P S50V T52D V53W C1C4 R47P S50E T52S V53W 6924F9 R47P S50G T52D 6881E11 R47P S50N T52C 6868F10 R47P T52S 6883G10 R47P S50D T52S 6939G4 R47P S50C T52D 11463D8 R47P S50F T52D 9667A11 R47N S50N T52D V53A 9675C8 R47Y S50A T52D V53A 9650E5 R47N S50W T52G V53H 9875B9 R47N S50N T52D V53W 9862B9 R47D S50W T52G V53W 9728G11 R47N S50W T52G V53W 11461D8 R47F S50A T52D V53A 11461A2 R47P S50F T52D V53I

Example 2 Construction of Error Prone PCR Library

Mutants obtained in Example 1, with mutations in their cofactor binding sites which exhibited relatively good NADH activities, were used as the DNA template to prepare the error prone (ePCR) libraries using the GeneMorph II kit (Stratagene) as recommended by the manufacturer. All the epPCR libraries target the N-terminal (which contains the NADPH binding site) of PF5_KARI. The forward primer (SEQ ID No: 20) and the reverse primer (SEQ ID No: 22) were used for all ePCR libraries.

The DNA templates for the n^(th) epPCR library were mutants having good NADH activity from the (n−1)^(th) epPCR library. The templates of the first epPCR library were mutants having relatively good NADH activity from libraries N and O. The mutations rate of library made by this kit was controlled by the amount of template added in the reaction mixture and the number of amplification cycles. Typically, 1.0 ng of each DNA template was used in 100 μl of reaction mixture. The number of amplification cycles was 70. The following conditions were used for the PCR reaction: The starting temperature was 95° C. for 30 sec followed by 70 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 30 min, and 70° C. for 2 min. After the first 35 heating/cooling cycles finished, more dNTP and Mutazyme II DNA polymerase were added. The PCR product was cleaned up using a DNA cleaning kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer. The cleaned PCR product was treated as Megaprimer and introduced into the vector using the Quickchange kit as described in Example 1. Table 4 below lists the KARI mutants obtained and the significant improvement observed in their NADH binding ability. The K_(M) was reduced from 1100 μM for mutant C3B11 to 50 μM for mutant 12957G9.

TABLE 4 List of some mutants with their measured K_(M) values NADH Mutant Mutation Locations K_(M) (μM) C3B11 R47F/S50A/T52D/V53W 1100 SB3 R47G/S50W/T52G/V53W 500 11518B4 R47N/S50N/T52D/V53A/A156V 141 11281G2 R47N/S50N/T52D/V53A/A156V/L165M 130 12985F6 R47Y/S50A/T52D/V53A/L61F/A156V 100 13002D8 R47Y/S50A/T52D/V53A/L61F/A156V/G170A 68 12957G9 Y24F/R47Y/S50A/T52D/V53A/L61F/G170A 50 12978D9 R47Y/S50A/T52D/V53A/L61F/Q115L/A156V 114

Example 3 Identification of Amino Acids for Cofactor Specificity Switching Using Bioinformatic Tools

To discover if naturally existing KARI sequences could provide clues for amino acid positions that should be targeted for mutagenesis, multiple sequence alignment (MSA) using PF5_KARI, its close homolog PAO1_KARI and three KARI sequences with measureable NADH activity, i.e., B. Cereus ilvC1 and ilvC2 and spinach KARI were performed (FIG. 8). Based on the multiple sequence alignment, positions 33, 43, 59, 61, 71, 80, 101, and 119 were chosen for saturation mutagenesis. Saturation mutagenesis on all of these positions was performed simultaneously using the QuickChange II XL site directed mutagenesis kit (Catalog #200524, Stratagene, La Jolla Calif.) with the manufacturer's suggested protocol. Starting material for this mutagenesis was a mixed template consisting of the mutants already identified in Example 2, Table 4. The primers used are listed in Table 5. The library of mutants thus obtained were named “library Z”. Mutants with good NADH activity from this library were identified using high throughput screening and their KARI activity and the K_(M) for NADH were measured as described above. These mutants (Table 6) possess much lower K_(M) s for NADH compared to the parent templates (Table 4). A Megaprimer, using primers (SEQ ID Nos. 20 and 58), was created and mutations at positions 156 and 170 were eliminated. Further screening of this set of mutants identified mutant 3361G8 (SEQ ID NO: 67) (Table 7). The hits from library Z were further subjected to saturation mutagenesis at position 53 using primers (SEQ ID Nos. 20 and 21), and subsequent screening identified the remaining mutants in Table 7. As shown in Table 7 the new mutants possessed much lower K_(M) for NADH (e.g., 4.0 to 5.5 μM) compared to mutants listed in Table 6 (e.g., 14-40 μM).

TABLE 5 Primers for Example 5 Targeted position(s) of Pf5_ilvC Primers  33 pBAD-405-C33_090808f: GCTCAAGCANNKAACCTGAAGG (SEQ ID NO: 49) pBAD-427-C33_090808r: CCTTCAGGTTKNNTGCTTGAGC (SEQ ID NO: 50)  43 pBAD-435-T43_090808f: GTAGACGTGNNKGTTGGCCTG (SEQ ID NO: 51) pBAD-456-T43_090808r: CAGGCCAACKNNCACGTCTAC (SEQ ID NO: 52)  59 and 61 pBAD-484-H59L61_090808f: CTGAAGCCNNKGGCNNKAAAGTGAC (SEQ ID NO: 53) pBAD-509-H59L61_090808r: GTCACTTTKNNGCCKNNGGCTTCAG (SEQ ID NO: 54)  71 pBAD-519-A71_090808f: GCAGCCGTTNNKGGTGCCGACT (SEQ ID NO: 55) pBAD-541-A71_090808r: AGTCGGCACCKNNAACGGCTGC (SEQ ID NO: 56)  80 pBAD-545-T80_090808f: CATGATCCTGNNKCCGGACGAG (SEQ ID NO: 57) pBAD-567-T80_090808r: CTCGTCCGGKNNCAGGATCATG (SEQ ID NO: 58) 101 pBAD-608-A101_090808f: CAAGAAGGGCNNKACTCTGGCCT (SEQ ID NO: 59) pBAD-631-A101_090808r: AGGCCAGAGTKNNGCCCTTCTTG (SEQ ID NO: 60) 119 pBAD-663-R119_090808f: GTTGTGCCTNNKGCCGACCTCG (SEQ ID NO: 61) pBAD-685-R119_090808r: CGAGGTCGGCKNNAGGCACAAC (SEQ ID NO: 62)

TABLE 6 List of some mutants with their measured K_(M) values (positions to be mutated in this library were indentified by bioinformatic tools) NADH Mutant Mutation Locations K_(M) (μM) ZB1 Y24F/R47Y/S50A/T52D/V53A/L61F/A156V 40 (SEQ ID NO: 24) ZF3 Y24F/C33L/R47Y/S50A/T52D/V53A/L61F 21 (SEQ ID NO: 25) ZF2 Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/A156V 17 (SEQ ID NO: 26) ZB3 Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/G170A 17 (SEQ ID NO: 27) Z4B8 C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V 14 (SEQ ID NO: 28)

TABLE 7 Mutants further optimized for improved K_(M) (for NADH) NADH K_(m) Mutant Mutation Locations (μM) 3361G8 C33L/R47Y/S50A/T52D/V53A/L61F/T80I 5.5 (SEQ ID NO: 67) 2H10 Y24F/C33L/R47Y/S50A/T52D/V53I/L61F/T80I/ 5.3 A156V (SEQ ID NO: 68) 1D2 Y24F/R47Y/S50A/T52D/V53A/L61F/T80I/ 4.1 A156V (SEQ ID NO: 69) 3F12 Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/T80I/ 4.0 A156V (SEQ ID NO: 70) 3361E1 Y24F/R47Y/S50A/T52D/V53I/L61F 4.5 (SEQ ID NO: 84)

Further analyses using bioinformatic tools were therefore performed to expand the mutational sites to other KARI sequences as described below.

Sequence Analysis

Members of the protein family of ketol-acid reducoisomorase (KARI) were identified through BlastP searches of publicly available databases using amino acid sequence of Pseudomonas fluorescens PF5 KARI (SEQ ID NO:17) with the following search parameters: E value=10, word size=3, Matrix=Blosum62, and Gap opening=11 and gap extension=1, E value cutoff of 10⁻³. Identical sequences and sequences that were shorter than 260 amino acids were removed. In addition, sequences that lack the typical GxGXX(G/A) motif involved in the binding of NAD(P)H in the N-terminal domain were also removed. These analyses resulted in a set of 692 KARI sequences.

A profile HMM was generated from the set of the experimentally verified Class I and Class II KARI enzymes from various sources as described in Table 8. Details on building, calibrating, and searching with this profile HMM are provided below. Any sequence that can be retrieved by HMM search using the profile HMM for KARI at E-value above 1E⁻³ is considered a member of the KARI family. Positions in a KARI sequence aligned to the following in the profile HMM nodes (defined below in the section of profile HMM building) are claimed to be responsible for NADH utilization: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 (the numbering is based on the sequences of Pseudomonas fluorescens PF5 KARI).

Preparation of Profile HMM

A group of KARI sequences were expressed in E. coli and have been verified to have KARI activity These KARIs are listed in Table 6. The amino acid sequences of these experimentally verified functional KARIs were analyzed using the HMMER software package (The theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., J. Mol. Biol. 235:1501-1531, 1994), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.). The output of the HMMER software program is a profile Hidden Markov Model (profile HMM) that characterizes the input sequences. As stated in the user guide, profile HMMs are statistical descriptions of the consensus of a multiple sequence alignment. They use position-specific scores for amino acids (or nucleotides) and position specific scores for opening and extending an insertion or deletion. Compared to other profile based methods, HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.).

The profile HMM was built as follows:

Step 1. Build a Sequence Alignment

The 25 sequences for the functionally verified KARIs listed above were aligned using Clustal W (Thompson, J. D., Higgins, D. G., and Gibson T. J., Nuc. Acid Res. 22: 4673 4680, 1994) with default parameters. The alignment is shown in FIG. 9.

TABLE 8 25 Experimentally verified KARI enzymes GI Number Accession SEQ ID NO: Microorganism 70732562 YP_262325.1 17 Pseudomonas fluorescens Pf-5 15897495 NP_342100.1 13 Sulfolobus solfataricus P2 18313972 NP_560639.1 14 Pyrobaculum aerophilum str. IM2 76801743 YP_326751.1 30 Natronomonas pharaonis DSM 2160 16079881 NP_390707.1 31 Bacillus subtilis subsp. subtilis str. 168 19552493 NP_600495.1 32 Corynebacterium glutamicum ATCC 13032 6225553 O32414 33 Phaeospririlum molischianum 17546794 NP_520196.1 15 Ralstonia solanacearum GMI1000 56552037 YP_162876.1 34 Zymomonas mobilis subsp. mobilis ZM4 114319705 YP_741388.1 35 Alkalilimnicola ehrlichei MLHE-1 57240359 ZP_00368308.1 36 Campylobacter lari RM2100 120553816 YP_958167.1 37 Marinobacter aquaeolei VT8 71065099 YP_263826.1 38 Psychrobacter arcticus 273-4 83648555 YP_436990.1 39 Hahella chejuensis KCTC 2396 74318007 YP_315747.1 40 Thiobacillus denitrificans ATCC 25259 67159493 ZP_00420011.1 41 Azotobacter vinelandii AvOP 66044103 YP_233944.1 42 Pseudomonas syringae pv. syringae B728a 28868203 NP_790822.1 43 Pseudomonas syringae pv. tomato str. DC3000 26991362 NP_746787.1 44 Pseudomonas putida KT2440 104783656 YP_610154.1 45 Pseudomonas entomophila L48 146306044 YP_001186509.1 46 Pseudomonas mendocina ymp 15599888 NP_253382.1 16 Pseudomonas aeruginosa PAO1 42780593 NP_977840.1 47 Bacillus cereus ATCC 10987 42781005 NP_978252.1 48 Bacillus cereus ATCC 10987 266346 Q01292 18 Spinacia oleracea Step 2. Build a Profile HMM

The hmmbuild program was run on the set of aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new profile HMM, and saves the profile HMM to file. Using this program an un-calibrated profile was generated from the multiple sequence alignment for twenty-four experimentally verified KARIs as described above.

The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a profile HMM. A profile HMM is a linear state machine consisting of a series of nodes, each of which corresponds roughly to a position (column) in the multiple sequence alignment from which it is built. If gaps are ignored, the correspondence is exact, i.e., the profile HMM has a node for each column in the alignment, and each node can exist in one state, a match state. The word “match” here implies that there is a position in the model for every position in the sequence to be aligned to the model. Gaps are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”.

A profile HMM has several types of probabilities associated with it. One type is the transition probability—the probability of transitioning from one state to another. There are also emissions probabilities associated with each match state, based on the probability of a given residue existing at that position in the alignment. For example, for a fairly well-conserved column in an alignment, the emissions probability for the most common amino acid may be 0.81, while for each of the other 19 amino acids it may be 0.01.

A profile HMM is completely described in a HMMER2 profile save file, which contains all the probabilities that are used to parameterize the HMM. The emission probabilities of a match state or an insert state are stored as log-odds ratio relative to a null model: log₂ (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24. State transition scores are also stored as log odds parameters and are proportional to log₂(t_x). Where t_x is the transition probability of transiting from one state to another state.

Step 3. Calibrate the Profile HMM

The profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. Hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for the profile HMM.

The calibrated profile HMM for the set of KARI sequences is provided appended hereto as a profile HMM Excel chart (Table 9). In the main model section starting from the HMM flag line, the model has three lines per node, for M nodes (where M is the number of match states, as given by the LENG line). The first line reports the match emission log-odds scores: the log-odds ratio of emitting each amino acid from that state and from the Null model. The first number if the node number (1 . . . M). The next K numbers for match emission scores, one per amino acid. The highest scoring amino acid is indicated in the parenthesis after the node number. These log-odds scores can be converted back to HMM probabilities using the null model probability. The last number on the line represents the alignment column index for this match state. The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E.

Step 4. Test the Specificity and Sensitivity of the Built Profile HMMs

The Profile HMM was evaluated using hmmsearch, which reads a Profile HMM from hmmfile and searches a sequence file for significantly similar sequence matches. The sequence file searched contained 692 sequences (see above). During the search, the size of the database (Z parameter) was set to 1 billion. This size setting ensures that significant E-values against the current database will remain significant in the foreseeable future. The E-value cutoff was set at 10.

An hmmersearch, using hmmsearch, with the profile HMM generated from the alignment of the twenty-five KARIs with experimentally verified function, matched all 692 sequences with an E value <10⁻³. This result indicates that members of the KARI family share significant sequence similarity. A hmmersearch with a cutoff of E value 10⁻³ was used to separate KARIs from other proteins.

Step 5. Identify Positions that are Relevant for NAD(P)H Utilization.

Eleven positions have been identified in KARI of Pseudomonas fluorescens Pf-5 that switches the cofactor from NADPH to NADH. Since the KARI sequences share significant sequence similarity (as described above), it can be reasoned that the homologous positions in the alignment of KARI sequences should contribute to the same functional specificity. The profile HMM for KARI enzymes has been generated from the multiple sequence alignment which contains the sequence of Pseudomonas fluorescens Pf-5 KARI. The eleven positions in the profile HMM representing the columns in the alignment which correspond to the eleven cofactor switching positions in Pseudomonas fluorescens Pf-5 KARI are identified as positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170. The lines corresponding to these positions in the model file are highlighted in yellow in Table 9.

For any query sequence, hmm search is used to search the profile HMM for KARI against the query sequence and the alignment of the query to the HMM is recorded in the output file. In the alignment section of the output, the top line is the HMM consensus. The amino acid shown for the consensus is the highest probability amino acid at that position according to the HMM (not necessarily the highest scoring amino acid). The center line shows letters for “exact” matches to the highest probability residue in the HMM, or a “+” when the match has a positive score. The third line shows the sequence itself. The positions in the query sequence that are deemed as relevant for cofactor switching are identified as those that are aligned to these eleven nodes in the profile HMM as described above. An example of the alignment of Pseudomonas fluorescens Pf-5 KARI to the profile HMM of KARI is shown in FIG. 10 and the eleven positions that are responsible for cofactor switching are shaded in grey.

Example 4 Construction of a Site-Saturation Gene Library for Complete Cofactor Switching to NADH

To construct the site-saturation gene library for KARI mutants, mutants 3361E1, 3361G8, 1D2, 2H10, 3F12, & Z4B8 (see Example 3, Tables 6 and 7) were used as templates. The library was constructed using QuickChange kit (Cat#200524, Stratagene, La Jolla, Calif.). The concentration of each mutant in the template mixture was 5.0 ng/μl. The two primers (2.5 nM) introducing saturation mutagenesis at positions 47, 50, 52 and 53, were PF5_(—)4Mt111008.f (SEQ ID NO: 71) and PF5_(—)4Mt111008.r (SEQ ID NO: 72).

The PCR Reaction Mixture Contained:

10 × reaction buffer 5.0 μl PF5_4Mt111008.f 2.0 μl PF5_4Mt111008.r 2.0 μl 50 x dNTP 1.0 μl DNA Template 1.0 μl PfuUltra 1.0 μl Water  38 μl The PCR Reaction Program was:

1) 95° C.  30 sec 2) 95° C.  30 sec 3) 55° C. 1.0 min 4) 68° C. 6.0 min 5) Go to step (2) Repeat 35 times 6) 68° C. 8.0 min 7) 4° C. press Enter

The mixture was placed in a thin well 200 μl tube for the PCR reaction in a Mastercycler gradient equipment (Brinkmann Instruments, Inc. Westbury, N.Y.). After the PCR reaction, 1.0 μl Dpn I restriction enzyme (supplied with the kit above) was directly added into the PCR reaction mixture, which was then incubated at 37° C. for 1 h to remove the DNA templates. The Dpn I digested PCR product was cleaned up by the Zymo DNA clearance kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer.

The cleaned PCR product was transformed into an electrocompetent strain of E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, Calif.). The transformed clones were streaked on agar plates containing the LB medium and 100 μg/ml ampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Dozens of clones were randomly chosen for DNA sequencing to confirm the quality of the library. Several mutants identified in this library (Table 10 and FIG. 11) had very low NADPH activity while they had good NADH activity. Their cofactor consumption is listed in Table 11 (The data was based on three parallel measurements). “Negative” in the following Tables refers to an empty pBAD vector without the KARI gene.

TABLE 10 List of some of the mutants identified in Example 1 Mutant Mutation Locations JB1C6 Y24F/C33L/R47H/S50D/T52Y/V53Y/L61F/T80I/A156V 16445E4 C33L/R47P/S50V/T52D/V53G/L61F/T80I/A156V 16468D7 Y24F/C33L/R47T/S50I/T52D/V53R/L61F/T80I/A156V 16469F3 C33L/R47E/S50A/T52D/V53A/L61F/T80I

TABLE 11 The cofactor consumption of some mutants following a 5 min reaction (decrease in OD₃₄₀ nm) 0.2 mM 0.2 mM NADH NADPH Mutants average stdev average stdev JB1C6 −0.232 0.127 −0.019 0.009 16445E4 −0.152 0.057 −0.013 0.001 16468D7 −0.153 0.012 −0.039 0.020 16469F3 −0.054 0.069 −0.025 0.016 Z4B8 −0.178 0.042 −0.170 0.013 PF5_WT −0.078 0.014 −0.320 0.024 Negative −0.061 0.029 −0.015 0.014

Example 5 Construction of a Domain Swapping Library

In this Example the beneficial mutations outside the cofactor binding sites and the beneficial mutations within the cofactor binding sites were combined to create a domain swapping library.

Mutants, which had mutations in the cofactor binding site and exhibited only NADH activity (SE1, SB3, SE2, SD3, C2F6, C3B11, C4D12, 9650E5, 9667A11, 9862B9, 9875B9, 11461D8, 11463D8, 11518B4, SEQ ID NOs: 85-98), were used to obtain additional beneficial mutations in the cofactor binding site. Two primers, pBAD_(—)230f (SEQ ID NO: 73) and pBAD_(—)601_(—)021308r (SEQ ID NO: 74), were used to amplify the mutants listed in Table 12. PCR reagents used were from Invitrogen (Cat#10572-014, Invitrogen, Carlsbad, Calif.).

The PCR Reaction Mixture Contained:

PCR SuperMix 180 μl  pBAD_230.f (18 nM) 5.0 μl pBAD_601_021308r (10 nM) 9.0 μl Template mix (5.0 ng/μl) 6.0 μl The PCR Reaction Program was:

(1) 95° C. 30 sec (2) 95° C. 20 sec (3) 55° C. 20 sec (4) 72° C. 60 sec (5) Go to step (2) repeat 35 times (6) 72° C.  4 min (7) 4° C. press enter

After the PCR reaction, 1.0 μl Dpn I restriction enzyme (supplied with the kit above) was directly added into the PCR reaction mixture, which was then incubated at 37° C. for 1 h to remove the DNA templates. The Dpn I digested PCR product was cleaned up by the Zymo DNA clearance kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer and 42 μl cleaned DNA product containing beneficial mutations in the cofactor binding sites obtained was designated as Megaprimer.

The Megaprimers thus obtained were then used to generate the domain swapping library using the QuickChange II XL site directed mutagenesis kit (Catalog #200524, Stratagene, La Jolla Calif.). The templates used in Example 4 were also used in this experiment. A 50 μl reaction mixture containing: 5.0 μl of 10× reaction buffer, 1.0 μl of 5.0 ng/μl template, 42 μl Megaprimer, 1.0 μl of 40 mM dNTP mix, 1.0 μl pfu-ultra DNA polymerase was prepared. Except for the Megaprimer and the templates, all reagents used here were supplied with the purchased kit. This reaction mixture was placed in a thin well 200 μl-capacity PCR tube and the following reactions were used for the PCR. The starting temperature was 95° C. for 30 sec followed by 30 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 1 min, and 68° C. for 6 min. At the completion of the temperature cycling, the samples were kept at 68° C. for 8 min, and then stored at 4° C. for later processing. Dpn I restriction enzyme (1.0 μl) (supplied with the kit above) was directly added to the finished reaction mixture, enzyme digestion was performed at 37° C. for 1 h and the PCR product was cleaned up using a DNA cleaning kit (Zymo Research). The cleaned PCR product (10 μl) contained mutated genes for a gene library.

The mutated genes were transformed into an electrocompetent strain of E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, Calif.). The transformed clones were streaked on LB agar plates containing 100 μg/ml ampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Dozens of clones were randomly chosen for DNA sequencing to confirm the quality of the library.

This library yielded many mutants with high NADH activity (low K_(M) for NADH), which also had very low NADPH activity. (Table 12 and FIG. 12). Their cofactor consumption is also shown in Table 13 (The data was based on three parallel measurements).

TABLE 12 Mutants with improved K_(M) (for NADH) obtained from the domain swapping library NADH K_(M) Mutant Mutation Locations (μM) JEA1 Y24F/C33L/R47P/S50F/T52D/L61F/T80I/A156V 9.1 JEG2 Y24F/C33L/R47F/S50A/T52D/V53A/L61F/T80I/ 9.4 A156V JEG4 Y24F/C33L/R47N/S50N/T52D/V53A/L61F/T80I/ 9.6 A156V JEA7 Y24F/C33L/R47P/S50N/T52D/V53A/L61F/T80I/ 10.6 A156V JED1 C33L/R47N/S50N/T52D/V53A/L61F/T80I/ 11.0 A156V

TABLE 13 The cofactor consumption of some mutants after 5 min reaction (decrease in OD₃₄₀ nm) 0.2 mM 0.2 mM NADH NADPH Mutants average stdev average stdev JEA1 −0.285 0.030 −0.110 0.025 JED1 −0.287 0.032 −0.074 0.014 JEG2 −0.261 0.009 −0.078 0.009 JEG4 −0.227 0.016 −0.050 0.016 JEA7 −0.205 0.079 −0.038 0.009 Z4B8 −0.178 0.042 −0.170 0.013 PF5_WT −0.078 0.014 −0.320 0.024 Negative −0.061 0.029 −0.015 0.014

Example 6 Thermostability of PF5-ILVC and its Mutants

The wildtype PF5-ILVC and various cells containing mutated pBad-ilvC were grown overnight at 37° C. in 25 ml of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v) arabinose inducer while shaking at 250 rpm. The cells were then harvested by centrifugation at 18,000×g for 1 min at room temperature and the cell pellets were re-suspended in 300 μl of BugBuster Master Mix (EMD Chemicals). The reaction mixture was first incubated at room temperature for 20 min and aliquots of this cell mixture (e.g. 50 μl) were incubated at different temperatures (from room temperature to 75° C.) for 10 min. The precipitate was removed by centrifugation at 18,000×g for 5 min at room temperature. The remaining activity of the supernatant was analyzed as described above. As shown in FIG. 7, pBad-ilvC was very stable with T₅₀ at 68° C. (T₅₀ is the temperature, at which 50% of protein lost its activity after 10 min incubation).

The thermostability of PF5-ilvC allowed destruction of most of the other non-KARI NADH oxidation activity within these cells, reducing the NADH background consumption and thus facilitating the KARI activity assays. This heat treatment protocol was used in all screening and re-screening assays. The mutants thus obtained were all thermostable which allowed easier selection of the desirable mutants.

Example 7 Stoichiometric Production of 2,3-Dihydroxyisovalerate by KARI During Consumption of NADH or NADPH as Cofactors

Screening and routine assays of KARI activity rely on the 340 nm absorption decrease associated with oxidation of the pyridine nucleotides NADPH or NADH. To insure that this metric was coupled to the formation of the reaction product (i.e., 2,3-dihydroxyisovalerate), oxidation of both pyridine nucleotide and formation of 2,3-dihydroxyisovalerate were measured in the same samples.

The oxidation of NADH or NADPH was measured at 340 nm in a 1 cm path length cuvette on a Agilent model 8453 spectrophotometer (Agilent Technologies, Wilmington Del.). Crude cell extract (0.1 ml) prepared as described above containing either wild type PF5 KARI or the C3B11 mutant, was added to 0.9 ml of K-phosphate buffer (10 mM, pH 7.6), containing 10 mM MgCl₂, and 0.2 mM of either NADPH or NADH. The reaction was initiated by the addition of acetolactate to a final concentration of 0.4 mM. After 10-20% decrease in the absorption (about 5 min), 50 μl of the reaction mixture was rapidly withdrawn and added to a 1.5 ml Eppendorf tube containing 10 μl 0.5 mM EDTA to stop the reaction and the actual absorption decrease for each sample was accurately recorded. Production of 2,3-dihydroxyisovalerate was measured and quantitated by HPLC/MS as described above.

The coupling ratio is defined by the ratio between the amount of 2,3-dihydroxyisovalerate (DHIV) produced and the amount of either NADH or NADPH consumed during the experiment. The coupling ratio for the wild type enzyme (PF5-ilvC), using NADPH, was 0.98 DHIV/NADPH, while that for the mutant (C3B11), using NADH, was on average around 1.10 DHIV/NADPH underlining the high activity of the mutant enzyme to consume NADH and produce DHIV.

TABLE 9 HMMER2.0 [2.2g] File format version: a unique identifier for this save file format. NAME Functionally Verified KARls Name of the profile HMM LENG 354 Mode length: the number of match states in the model. ALPH Amino Symbol alphabet: This determines the symbol alphabet and the size of the symbol emission probability distributions. Amino, the alphabet size Is set to 20 and the symbol alphabet to “ACDEFGHIKLMNPQRSTVWY” (alphabetic order). MAP yes Map annotation flag: If set to yes, each line of data for the match state/consensus column in the main section of the file is followed by an extra number. This number gives the index of the alignment column that the match state was made from. This information provides a “map” of the match states (1...M) onto the columns of the alignment (1.alen). It is used for quickly aligning the model back to the original alignment, e.g. when using hmmalign-mapali. COM hmmbuild-n Functionally Verified KARls exp-KARl.hmm exp-KARl_mod.aln Command line for every HMMER command that modifies the save file: This one means that hmmbuild (default patrameters) was applied to generate the save file. COM hmmcalibrate exp-KARl.hmm Command line for every HMMER command that modifies the save file: This one means that hmmcalibrate (default parametrs) was applied to the save profile. NSEQ 25 Sequence number: the number of sequences the HMM was trained on DATE Mon Dec. 8 17:34:51 2008 Creation date: When was the save file was generated. XT-8455-4-1000-1000-8455-4-8455-4 Eight “special” transitions for controlling parts of the algorithm-specific parts of the Plan7 model. The null probability used to convert these back to model probabilities is 1.0. The order of the eight field is N->B, N->N, E->C, E->J, C->T, C->C, J->B, J->J. NULT-4-8455 The transition probability distribution for the null model (single G state). NULE 595-1558 85336-294-453-1158 197 249 902-1085-142-21-313 45 531 201 384-1998-644 The extreme value distribution parameters μ and lambda respectively; both floating point values. EVD-333.712708 0.110102 These values are set when the model is calibrated with hmmcalibrate. They are used to determine E-values of bit scores. 

What is claimed is:
 1. A recombinant mutant ketol-acid reductoisomerase enzyme having the amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 67, 68, 70, 75, 79, 80, 81 and 82 having ketol-acid reductoisomerase activity.
 2. A method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising: a) providing a mutant enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 67, 68, 69, 70, and 84; b) constructing a site-saturation library targeting amino acid positions 47, 50, 52 and 53 of the mutant enzyme of (a); and c) screening the site-saturation library of (b) to identify mutants which accept NADH instead of NADPH as cofactor.
 3. The method of claim 2 wherein the K_(M) for NADH is less than 15 μM.
 4. A recombinant mutant ketol-acid reductoisomerase enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOS: 75, 76, 77 and 78 and having ketol-acid reductoisomerase activity.
 5. A recombinant mutant ketol-acid reductoisomerase enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOS: 79, 80, 81, 82, and 83 and having ketol-acid reductoisomerase activity.
 6. The mutant ketol-acid reductoisomerase enzyme of claim 5 having the amino acid sequence of SEQ ID NO:
 79. 